CN117835991A

CN117835991A - Chimeric costimulatory receptors, chemokine receptors and their use in cellular immunotherapy

Info

Publication number: CN117835991A
Application number: CN202280043474.7A
Authority: CN
Inventors: R·库巴斯; F·G·福格特; 张永良
Original assignee: Iovance Biotherapeutics Inc
Current assignee: Iovance Biotherapeutics Inc
Priority date: 2021-04-19
Filing date: 2022-04-19
Publication date: 2024-04-05

Abstract

The present invention provides compositions comprising chimeric receptors, including chimeric co-stimulatory receptors (CCR), and/or chemokine receptors, methods for preparing CCR and/or chemokine receptors, and therapeutic populations of tumor-infiltrating lymphocytes, bone marrow-infiltrating lymphocytes, and peripheral blood lymphocytes that express CCR and/or chemokine receptors, having increased therapeutic efficacy for treating cancers, including solid tumor cancers, and other advantages.

Description

Chimeric costimulatory receptors, chemokine receptors and their use in cellular immunotherapy

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/176,675 (2021, 4, 19, application), U.S. provisional patent application No. 63/223,925 (2021, 7, 20, application), U.S. provisional patent application No. 63/254,297 (2021, 10, 11, application), and U.S. provisional patent application No. 63/284,177 (2021, 11, 30, application), the disclosures of each of which are incorporated herein by reference in their entirety.

Background

Treatment of solid tumor cancers remains challenging, particularly for patients who do not respond to common initial on-line therapies, including chemotherapy, targeted therapies, and checkpoint inhibitors, such as nivoruniab (nivolumab), pembrolizumab (pembrolizumab), ipilimab (ipilimumaab), atuzumab (atezolizumab), avalimab (avelumab), devaluzumab (durvalumab), and therapies using a combination of these immunotherapies (e.g., nivoruzumab and ipilimab) or a combination of these immunotherapies with chemotherapy. Treatment of large (bulk), refractory cancers with adoptive transfer Tumor Infiltrating Lymphocytes (TILs) represents a powerful treatment regimen for patients with solid tumor cancers that are ill-prognosis, including those who failed initial on-line therapy. Gattineni et al, nat. Rev. Immunol.2006,6,383-393. Successful immunotherapy requires a large amount of active TIL, whereas commercialization requires a robust and reliable process. This is a challenge to achieve due to technical, logistical and regulatory issues of cell expansion. IL-2 based TIL amplification and the subsequent rapid amplification process (REP) have become the preferred methods for TIL amplification due to their speed and efficiency. Dudley et al, science 2002,298,850-54; dudley et al, J.Clin.Oncol.2005,23,2346-57; dudley et al, J.Clin.Oncol.2008,26,5233-39; riddell et al, science 1992,257,238-41; dudley et al, J.Immunother.2003,26,332-42.REP can lead to 1,000-fold TIL expansion over a 14 day period, although it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as Monocytes (MNCs)) typically from multiple donors as feeder cells, as well as anti-CD 3 antibody (OKT 3) and high doses of IL-2.Dudley et al, J.Immunother.2003,26,332-42. TIL subjected to REP procedures has produced successful adoptive cell therapies in both anterior and posterior therapeutic settings following host immunosuppression in melanoma, head and neck cancer, non-small cell lung cancer and cervical cancer patients. Jimeno et al, master 353,SITC Annual Meeting,Nov.9-14,2020; sarnaik et al, oral Presentation at ASCO Annual Meeting, may 29-30,2020; jazaeri et al, master 182,ASCO Annual Meeting,May 31-June 4,2019.

Chimeric co-stimulatory receptors (CCR) are genetically engineered chimeric receptors designed to provide co-stimulatory signals to effector cells (e.g., T cells) to enhance activation. Sadelain et al, cancer Discovery,2013,3,388-398; liao et al, biomaker res.2020,8,57.CCR are most often associated with T cell therapies based on Chimeric Antigen Receptor (CAR) modified T cell (CAR-T) products, which can be combined with CARs to enhance activation. However, CCR has not been extensively explored with other emerging cell therapies, including polyclonal TIL therapies in solid tumor cancers, such as activation with tumor-associated antigens or other antigens. Although TIL therapies have shown safety and efficacy at present, particularly in patients with locally advanced or metastatic solid tumor cancers, there is a significant unmet need for TIL therapies with other factors that improve efficacy, duration of response, and safety.

Disclosure of Invention

The present invention provides methods of making and using TILs and compositions thereof for treating solid tumor patients, the TILs having enhanced properties using CCR.

In some embodiments, the invention includes a method of treating cancer by administering to a patient in need of treatment a tumor-infiltrating lymphocyte (TIL), a bone marrow-infiltrating lymphocyte (MILs), or a population of Peripheral Blood Lymphocytes (PBLs), wherein the TIL, MILs, or PBLs are genetically modified to express a chimeric co-stimulatory receptor (CCR), the CCR comprising:

i. An extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

wherein the cancer is treated by administering a population of TILs, the method comprising:

(a) Obtaining and/or receiving a first population of TILs from a tumor resected from a patient by treating a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests;

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

(c) Performing a first expansion by culturing a first population of TILs in a first cell culture medium comprising IL-2 and optionally OKT-3 antibodies and Antigen Presenting Cells (APCs), resulting in 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 a second population of TILs, the transition from step (b) to step (c) occurring without opening the system;

(d) Genetically modifying the second TIL population to express CCR;

(e) Performing a second amplification of the second TIL population in a second cell culture medium comprising IL-2, OKT-3 antibodies and APC to produce a third TIL population; wherein the second amplification is performed for about 3 to 14 days to obtain a third population of TILs, the third population of TILs being a population of therapeutic TILs, the second amplification being performed in a closed container providing a second gas-permeable surface area;

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

(g) Transferring the collected TIL population from step (f) to an infusion bag, wherein the transfer of step (e) to step (f) occurs without opening the system;

(h) Cryopreserving the infusion bag from step (f) containing the collected TIL population using a cryopreservation process; and

(i) Administering to the patient a therapeutically effective dose of the third TIL population from the infusion bag of step (g).

i. An extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

(i) Administering to the patient a therapeutically effective dose of a third population of TILs from the infusion bag of step (g);

wherein the extracellular domain comprises an scFv binding domain.

i. An extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

wherein the extracellular domain comprises an scFv binding domain that binds to a protein selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, fra, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, IL-13 ra 2, FAS, tgfbrii, and MUC 16.

i. An extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

wherein the extracellular domain is a PD-1 domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

wherein the intracellular domain is selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

wherein the intracellular domain is selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof, and the transmembrane domain is selected from the group consisting of the transmembrane regions of CD3 α, CD3 β, CDζ, CD3 ε, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2Rα, IL-2Rβ, and IL-2Rγ.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

wherein the intracellular domain is selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof, and the transmembrane domain is selected from the group consisting of the transmembrane regions of CD3 α, CD3 β, CDζ, CD3 ε, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2Rα, IL-2Rβ, and IL-2Rγ, and combinations thereof, and step (d) further comprises modifying TIL using the lentiviral gene to express CCR.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

wherein the intracellular domain is selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof, the transmembrane domain is selected from the group consisting of CD3 α, CD3 β, CD ζ, CD3 ε, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2Rα, IL-2Rβ, and IL-2Rγ, and combinations thereof, and step (d) further comprises modifying the TIL, MIL or PBL with a lentiviral gene to express CCR, the TIL, MIL or PBL being further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), SOCS1, ANKRD11, BCOR, and combinations thereof.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

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

(d) Genetically modifying the second TIL population to express CCR;

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

wherein the intracellular domain is selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, and combinations thereof, the transmembrane domain is selected from the group consisting of CD3 α, CD3 β, CD ζ, CD3 ε, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2Rα, IL-2Rβ, and IL-2Rγ, and combinations thereof, and step (d) further comprises modifying the TIL, MIL or PBL with a lentiviral gene to express CCR, the TIL, MIL or PBL being further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), SOCS1, ANKRD11, BCOR, and combinations thereof, which is a solid tumor cancer treated by administration of TIL.

In any of the foregoing embodiments, the cancer may be selected from sarcoma, pancreatic cancer, liver cancer, glioblastoma, gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, lung cancer, non-small cell lung cancer, mesothelioma, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell cancer, and the patient is a human.

In any of the foregoing embodiments, the cancer is further treated with a PD-1 inhibitor or a PD-L1 inhibitor in combination with TIL, wherein the PD-1 inhibitor or the PD-L1 inhibitor is selected from nivolumab, pembrolizumab, cetirilizumab (cemiplimab), tirelizumab (tislealizumab), melighur Li Shan antibody (sintilimab), terlipp Li Shan antibody (toripalimab), doslimab (dostarlimab), devaluzumab, ivermectin, atuzumab, retifanlimab (retifanlimab), and fragments, variants and biological analogs thereof.

In any of the foregoing embodiments, the cancer is non-small cell lung cancer, and the patient has at least one of:

1) Tumor fraction (TPS) <1% of pre-determined PD-L1;

2) TPS fraction of PD-L1 is 1% to 49%, or

3) The absence of more than one driving mutation was determined in advance.

In any of the preceding embodiments, the cancer is non-small cell lung cancer, and the TPS of PD-L1 in the patient is <1%.

In any of the foregoing embodiments, the patient has a cancer that is not amenable to treatment by: EGFR inhibitors, BRAF inhibitors, ALK inhibitors, C-Ros inhibitors, RET inhibitors, ERBB2 inhibitors, BRCA inhibitors, MAP2K1 inhibitors, PIK3CA inhibitors, CDKN2A inhibitors, PTEN inhibitors, UMD inhibitors, NRAS inhibitors, KRAS inhibitors, NF1 inhibitors, MET inhibitors, TP53 inhibitors, CREBBP inhibitors, KMT2C inhibitors, KMT2D mutations, ARID1A mutations, RB1 inhibitors, ATM inhibitors, SETD2 inhibitors, FLT3 inhibitors, PTPN11 inhibitors, FGFR1 inhibitors, EP300 inhibitors, MYC inhibitors, EZH2 inhibitors, JAK2 inhibitors, xw7 inhibitors, CCND3 inhibitors, and GNA11 inhibitors.

In any of the foregoing embodiments, the patient does not have one or more driving mutations, wherein the one or more driving mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20, KRAS mutations, BRAF V600 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 splice 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.

In any of the foregoing embodiments, the cancer exhibits a refractory or resistance to treatment by a chemotherapeutic agent or chemotherapy.

In any of the foregoing embodiments, the cancer exhibits refractory or resistant to treatment with Sub>A VEGF-Sub>A inhibitor, wherein the VEGF-Sub>A inhibitor is selected from bevacizumab (bevacizumab), lanbizumab (ranibizumab), ai Lusu mab (icrucumab), and fragments, variants and biological analogs thereof.

In any of the foregoing embodiments, the cancer exhibits refractory or resistant to treatment with a PD-1 inhibitor or a PD-L1 inhibitor, wherein the PD-1 inhibitor or the PD-L1 inhibitor is selected from nivolumab, pembrolizumab, cimepizumab, tirelimumab, singedi Li Shan antibody, terlipressin Li Shan antibody, doslimab, devaluzumab, eslimumab, alemtuzumab, remifur Li Shan antibody, and fragments, variants and biological analogs thereof.

In any of the foregoing embodiments, the cancer exhibits refractory or resistant to treatment with a CTLA-4 inhibitor, wherein the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab (tremelimumab), za Li Fu limumab (zalifrelimumab), and fragments, variants, and biological analogs thereof.

In any of the preceding embodiments, the IL-2 is initially present in the first cell culture medium and the second cell culture medium at an initial concentration of between 1000IU/mL and 6000 IU/mL.

In any of the preceding embodiments, the OKT-3 antibody is initially present in the second cell culture medium at an initial concentration of about 30 ng/mL.

In any of the preceding embodiments, the first 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 any of the preceding embodiments, the second 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 any of the foregoing embodiments, the method may further comprise the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third TIL population to the patient.

In any of the foregoing embodiments, the method may further comprise, prior to administering to the patientA step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to the third TIL population, the non-myeloablative lymphocyte depletion regimen comprising administering a dose of 60mg/m ² Cyclophosphamide/day for two days and then administered at a dose of 25mg/m ² Five days of fludarabine per day.

In any of the foregoing embodiments, the method may further comprise the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third TIL population to the patient, the non-myeloablative lymphocyte depletion regimen comprising administering a dose of 60mg/m ² Cyclophosphamide per day and dose 25mg/m ² Fludarabine/day for two days and subsequent administration at a dose of 25mg/m ² Three days of fludarabine per day.

In any of the foregoing embodiments, the method may further comprise the step of beginning treatment of the patient with the IL-2 regimen the next day after administration of the third TIL population to the patient.

In any of the foregoing embodiments, the method may further comprise the step of beginning treatment of the patient with the IL-2 regimen on the same day as the third TIL population is administered to the patient.

In any of the foregoing embodiments, the method can further comprise the step of treating the patient with an IL-2 regimen, wherein the IL-2 regimen is a high dose IL-2 regimen comprising administering 600,000 or 720,000IU/kg of the aldesleukin, or a fragment, variant or biological analog thereof, per eight hours at 15 minutes of bolus intravenous infusion until tolerizing.

In any of the foregoing embodiments, the method may further comprise the step of treating the patient with an IL-2 regimen, the IL-2 regimen comprising administration of Bei Jiade Lu Jin (bempeg alderslaukin) or a fragment, variant or biological analog thereof.

In any of the foregoing embodiments, the method can further comprise the step of treating the patient with an IL-2 regimen, the IL-2 regimen comprising administering THOR-707 or a fragment, variant or biological analog thereof.

In any of the foregoing embodiments, the method may further comprise the step of treating the patient with an IL-2 regimen, the IL-2 regimen comprising administering semw Lu Jin alpha (nemvaleukin alfa) or a fragment, variant or biological analog thereof.

In any of the foregoing embodiments, the method may further comprise the step of treating the patient with an IL-2 regimen, the IL-2 regimen comprising administering an antibody or fragment, variant or biological analog thereof, said antibody 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 any of the foregoing embodiments, a therapeutically effective population of TILs is administered, the therapeutically effective population of TILs comprising about 2 x 10 ⁹ Up to about 15X 10 ¹⁰ And TIL.

In any of the preceding embodiments, the first amplification is performed for a period of 11 days or less.

In any of the preceding embodiments, the second amplification is performed for a period of 11 days or less.

In some embodiments, the invention includes a composition comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs) genetically modified to express a chimeric co-stimulatory receptor (CCR), wherein the CCR comprises:

i. An extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

wherein the extracellular domain comprises an scFv binding domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

wherein the extracellular domain comprises an scFv binding domain selected from the group consisting of an anti-CD 19 domain, an anti-CD 20 domain, an anti-CD 22 domain, an anti-CD 24 domain, an anti-CD 33 domain, an anti-CD 38 domain, an anti-CD 39 domain, an anti-CD 73 domain, an anti-CD 123 domain, an anti-CD 138 domain, an anti-CD 228 domain, an anti-LRRC 15 domain, an anti-CEA domain, an anti-fra domain, an anti-EPCAM domain, an anti-PD-L1 domain, an anti-PSMA domain, an anti-gp 100 domain, an anti-MUC 1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD 2 domain, an anti-TROP-2 domain, an anti-GPC 3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-2 domain, an anti-MCM 5 domain, an anti-FAP domain, an anti-5T 4 domain, an anti-a-1 domain, an anti-B7-H3 domain, and an anti-MUC 16 domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

wherein the extracellular domain is a PD-1 domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

wherein the intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

wherein the intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof, and the transmembrane domain is selected from the group consisting of a CD3 α domain, a CD3 β domain, a CD ζ domain, a CD3 ε domain, a CD4 domain, a CD5 domain, a CD8 α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IL-1, an IgG domain, an IL-4R 2 domain, and an IL-2R 2 domain.

i. an extracellular domain;

an optional hinge domain;

an optional transmembrane domain; and

at least one intracellular domain;

wherein the intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof, the transmembrane domain is selected from the group consisting of a CD3 a domain, a CD3 β domain, a cdζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 a domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2 ra domain, an IL-2rβ domain, and an IL-2rγ domain, the TIL, MILs, or PBL being further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3) and combinations thereof.

In some embodiments, the invention includes a composition comprising a chimeric co-stimulatory receptor (CCR), wherein the CCR comprises:

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain.

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the extracellular protein domain comprises an scFv binding domain.

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the extracellular protein domain comprises an scFv binding domain selected from the group consisting of an anti-CD 19 domain, an anti-CD 20 domain, an anti-CD 22 domain, an anti-CD 24 domain, an anti-CD 33 domain, an anti-CD 38 domain, an anti-CD 39 domain, an anti-CD 73 domain, an anti-CD 123 domain, an anti-CD 138 domain, an anti-CD 228 domain, an anti-LRRC 15 domain, an anti-CEA domain, an anti-fra domain, an anti-EPCAM domain, an anti-PD-L1 domain, an anti-PSMA domain, an anti-gp 100 domain, an anti-MUC 1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD 2 domain, an anti-TROP-2 domain, an anti-GPC 3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-HER 2 domain, an anti-FAP domain, an anti-5T 4 domain, an anti-LFA-1 domain, an anti-B7-H3 domain, and an anti-MUC 16 domain.

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the extracellular protein domain is a PD-1 domain.

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof.

i. an extracellular protein domain;

Hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof, and the transmembrane protein domain is selected from the group consisting of a CD3 α domain, a CD3 β domain, a CD zeta domain, a CD3 ε domain, a CD4 domain, a CD5 domain, a CD8 α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IL-4 IgG domain, an IL-2R 2 domain, and an IgR 2 domain.

i. An extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof, the transmembrane protein domain is selected from the group consisting of a CD3 α domain, a CD3 β domain, a CD zeta domain, a CD3 ε domain, a CD4 domain, a CD5 domain, a CD8 α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154, an IL-4, an IgG domain, an IL-2R 2 domain, and an IgR 2 domain, the hinge protein domain is selected from the group consisting of a CD3 alpha domain, a CD3 beta domain, a CD zeta domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 alpha domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2R alpha domain, an IL-2R beta domain, and an IL-2R gamma domain.

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof, the transmembrane protein domain is selected from the group consisting of a CD3 α domain, a CD3 β domain, a CD zeta domain, a CD3 ε domain, a CD4 domain, a CD5 domain, a CD8 α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154, an IL-4, an IgG domain, an IL-2R 2 domain, and an IgR 2 domain, the hinge protein domain is selected from the group consisting of a CD3 a domain, a CD3 β domain, a cdζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 a domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2R a domain, an IL-2R β domain, and an IL-2R γ domain, and the composition further comprises tumor infiltrating lymphocytes.

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof, the transmembrane protein domain is selected from the group consisting of a CD3 α domain, a CD3 β domain, a CD zeta domain, a CD3 ε domain, a CD4 domain, a CD5 domain, a CD8 α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154, an IL-4, an IgG domain, an IL-2R 2 domain, and an IgR 2 domain, the hinge protein domain is selected from the group consisting of a CD3 a domain, a CD3 β domain, a cdζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 a domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2R a domain, an IL-2R β domain, and an IL-2R γ domain, and the composition further comprises bone marrow infiltrating lymphocytes.

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain;

wherein the intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof, the transmembrane protein domain is selected from the group consisting of a CD3 α domain, a CD3 β domain, a CD zeta domain, a CD3 ε domain, a CD4 domain, a CD5 domain, a CD8 α domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154, an IL-4, an IgG domain, an IL-2R 2 domain, and an IgR 2 domain, the hinge protein domain is selected from the group consisting of a CD3 alpha domain, a CD3 beta domain, a CD ζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 alpha domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2R alpha domain, an IL-2R beta domain, and an IL-2R gamma domain, and the composition further comprises peripheral blood lymphocytes.

In some embodiments, the invention includes a method of treating cancer by administering to a patient in need of treatment a tumor-infiltrating lymphocyte (TIL), a bone marrow-infiltrating lymphocyte (MILs), or a population of Peripheral Blood Lymphocytes (PBLs), wherein the TIL, MILs, or PBLs are genetically modified to express a chemokine receptor.

In some embodiments, the invention includes a method of treating cancer by administering to a patient in need of treatment a tumor-infiltrating lymphocyte (TIL), bone marrow-infiltrating lymphocyte (MILs), or Peripheral Blood Lymphocyte (PBL) population, wherein the TIL, MILs, or PBL is genetically modified to express a chemokine receptor, the cancer being treated by administering a population of TILs, the method comprising:

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

(d) Genetically modifying the second TIL population to express a chemokine receptor;

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

In some embodiments, the invention includes a method of treating cancer by administering to a patient in need of treatment a tumor-infiltrating lymphocyte (TIL), bone marrow-infiltrating lymphocyte (MILs), or Peripheral Blood Lymphocyte (PBL) population, wherein the TIL, MILs, or PBL is genetically modified to express a chemokine receptor, the cancer being treated by administering the TIL population, the method comprising, the chemokine receptor is a protein selected from CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof.

In some embodiments, the invention includes a method of treating cancer by administering to a patient in need of treatment a tumor-infiltrating lymphocyte (TIL), bone marrow-infiltrating lymphocyte (MILs), or Peripheral Blood Lymphocyte (PBL) population, the TIL, MILs, or PBL genetically modified to express a chemokine receptor, the cancer being treated by administering the TIL population, the method comprising, the chemokine receptor being a protein selected from CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof, and step (d) further comprises genetically modifying the TIL with a lentivirus or retrovirus to express the chemokine receptor.

In some embodiments, the invention includes a method of treating cancer by administering to a patient in need of treatment a population of tumor-infiltrating lymphocytes (TIL), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs), wherein the TIL, MILs, or PBLs are genetically modified to express a chemokine receptor, the cancer being treated by administering the population of TILs, the method comprising, the chemokine receptor being a protein selected from CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof, step (d) further comprising genetically modifying the TIL with a lentivirus or retrovirus to express a chemokine receptor, the TIL, MILs, or PBLs being further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), SOCS1, ANKRD11, BCOR, and combinations thereof.

In some embodiments, the invention includes a method of treating a cancer by administering to a patient in need of treatment a tumor-infiltrating lymphocyte (TIL), a bone marrow-infiltrating lymphocyte (MILs), or a population of Peripheral Blood Lymphocytes (PBLs), wherein the TIL, MILs, or PBLs are genetically modified to express a chemokine receptor, the cancer being a solid tumor cancer treated by administering the TIL.

In any of the foregoing embodiments, the cancer is further treated with a PD-1 inhibitor or a PD-L1 inhibitor in combination with TIL, wherein the PD-1 inhibitor or the PD-L1 inhibitor is selected from nivolumab, pembrolizumab, cimimab, tirelimumab, singedi Li Shan antibody, terlipressin Li Shan antibody, doslimab, devaluzumab, avalimab, alemtuzumab, remife Li Shan antibody, and fragments, variants, and biological analogs thereof.

1) Tumor fraction (TPS) <1% of pre-determined PD-L1;

2) TPS fraction of PD-L1 is 1% to 49%, or

3) The absence of more than one driving mutation was determined in advance.

In any of the foregoing embodiments, the cancer exhibits refractory or resistant to treatment with Sub>A VEGF-Sub>A inhibitor, wherein the VEGF-Sub>A inhibitor is selected from bevacizumab, lanbizumab, ai Lusu mab, and fragments, variants, and biological analogs thereof.

In any of the foregoing embodiments, the cancer exhibits refractory or resistant to treatment with a CTLA-4 inhibitor, wherein the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, za Li Fu limumab, and fragments, variants, and biological analogs thereof.

In any of the foregoing embodiments, the method may further comprise the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third TIL population to the patient, the non-myeloablative lymphocyte depletion regimen comprising administering a dose of 60mg/m ² Cyclophosphamide/day for two days and then administered at a dose of 25mg/m ² Five days of fludarabine per day.

In any of the foregoing embodiments, the method may further comprise the step of treating the patient with an IL-2 regimen, the IL-2 regimen comprising administration of Bei Jiade Lu Jin or a fragment, variant or biological analog thereof.

In any of the foregoing embodiments, the method may further comprise the step of treating the patient with an IL-2 regimen, the IL-2 regimen comprising administering inner tile Lu Jin alpha or a fragment, variant or biological analog thereof.

In some embodiments, the invention provides a composition comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs) genetically modified to express a chemokine receptor.

In some embodiments, the composition of the invention of claim 94, wherein said chemokine receptor is a protein selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof.

In some embodiments, the composition of any one of claims 94-95, wherein the TIL, MILs, or PBL is further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3) and combinations thereof.

In some embodiments, a composition is provided comprising a chemokine receptor, wherein the composition further comprises tumor-infiltrating lymphocytes, bone marrow-infiltrating lymphocytes, or peripheral blood lymphocytes.

Drawings

Fig. 1: the diagram of the exemplary generation 2 (process 2A) provides an overview of steps a through F.

Fig. 2A to 2C: process flow diagram for the 2 nd generation (process 2A) embodiment of TIL manufacture.

Fig. 3: a diagram of an embodiment of an exemplary manufacturing process for cryopreserving TIL (about 22 days) is shown.

Fig. 4: a diagram of an embodiment of process 2A (22 day TIL manufacturing process) is shown.

Fig. 5: comparison table of steps a through F for exemplary embodiments of process 1C and generation 2 (process 2A) for TIL manufacture.

Fig. 6: detailed comparison of process 1C for TIL manufacture with the implementation of generation 2 (process 2A).

Fig. 7: exemplary 3 rd generation TIL manufacturing process.

Fig. 8A to 8D: a) A comparison of the 2A process (about a 22 day process) and the 3 rd generation process embodiment (about a 14 day to 16 day process) for TIL manufacture is shown. B) The diagram of the exemplary 3 rd generation process provides an overview of steps a through F (about 14 to 16 day process). C) The figure provides an overview of each of three exemplary 3 rd generation processes and three process variations of steps a through F (about 14 to 16 day processes). D) An exemplary modified class 2 process provides an overview of steps a through F (about a 22 day process).

Fig. 9: experimental flow diagrams of the comparability between the 2 nd generation process (2A process) and the 3 rd generation process are provided.

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

Fig. 11: the table describes various features of embodiments of the 2 nd, 2.1 nd and 3.0 th generation processes.

Fig. 12: culture medium condition overview of the embodiment of the 3 rd generation procedure (referred to as 3.1 rd generation).

Fig. 13: the table describes various features of embodiments of the 2 nd, 2.1 nd and 3.0 th generation processes.

Fig. 14: the table compares various features of the embodiments of the 2 nd and 3.0 th generation processes.

Fig. 15: the table provides the media use for various embodiments of the amplification process.

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

Fig. 17: schematic diagram of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancy using a 3 rd generation expansion platform.

Fig. 18: structures I-A and I-B are provided. The cylinders refer to the respective 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 TNFRSF binding domains fold to form a trivalent protein that is then linked to a second trivalent protein by an IgG1-Fc (including CH3 and CH2 domains) that serves to link the two trivalent proteins together by disulfide bonds (small long oval), 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 linked by a linker _H And V _L A chain, which may comprise hydrophilic residues and Gly and Ser sequences providing softness, glu and Lys providing solubility.

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

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

Fig. 21: schematic of an exemplary embodiment of the 3.1 rd generation process (16 to 17 day process).

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

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

Fig. 24: schematic diagrams of exemplary embodiments of the 3 rd generation process (16 to 17 day process) show the preparation time axis.

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

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

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

Fig. 28: comparison of one embodiment of the 2 nd, 2.1 nd and 3 rd generation processes (16 day process).

Fig. 29: comparison of one embodiment of the 2 nd, 2.1 nd and 3 rd generation processes (16 day process).

Fig. 30: 3 rd generation embodiment components.

Fig. 31: flow chart comparison of the 3 rd generation embodiment (3.0 rd generation, 3.1 rd generation control, 3.1 rd generation test).

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

Fig. 33: a standard table is accepted.

Fig. 34: schematic diagram of exemplary scFv CCR constructs.

Fig. 35: exemplary PD-1 switch CCR design.

Fig. 36: exemplary PD-1 switch CCR designs with alternative CD28 signaling domains.

Fig. 37: exemplary CCR construct designs.

Fig. 38: anti-TROP-2 (V) comprising an IgG4 hinge and transmembrane domain and an IL-2Rβ intracellular domain _L -linker-V _H ) Exemplary vector designs and embodiments of the invention for lentiviral expression of CCR in TIL.

Fig. 39: anti-FAP-2 (V) comprising a CD8 alpha hinge and a transmembrane domain and an IL-2 Rbeta intracellular domain _L -linker-V _H ) Exemplary vector designs and embodiments of the invention for lentiviral expression of CCR in TIL.

Fig. 40: use of a polypeptide comprising a CD8 alpha hinge and a transmembrane domain and IL-2Ranti-PD-L1 (V) of 38A1 antibody to the beta intracellular domain _L -linker-V _H ) Exemplary vector designs and embodiments of the invention for lentiviral expression of CCR in TIL.

Fig. 41: exemplary vector designs for retroviral expression of CXCR1 in TIL and one embodiment of the invention.

Fig. 42: exemplary vector designs for retroviral expression of CCR8 in TIL and one embodiment of the present invention.

Fig. 43: flow cytometry analysis of cervical cancer tumor digests. EPCAM Phycoerythrin (PE)/TROP-2 PE.

Fig. 44: EPCAM of cervical cancer tumor digests (APC)/TROP-2 PE).

Fig. 45: EPCAM/TROP-2 expression on head and neck squamous cell carcinoma digests.

Fig. 46: EPCAM/TROP-2 expression on non-small cell lung cancer tumor digests.

Fig. 47: cell frequency distribution in TIL formulations of the 2 nd generation REP formulations. 9 different TILs were thawed and stained on different days for identification, PBMCs were used as control groups.

Fig. 48: cell frequency distribution in TIL formulations of the 2 nd generation REP formulations. 9 different TILs were thawed and stained on different days for identification, PBMCs were used as control groups.

Fig. 49: showing in CD8 ⁺ Flow cytometry results for chemokine receptors on TIL.

Fig. 50: showing the presence of CD4 ⁺ Flow cytometry results for chemokine receptors on TIL.

Fig. 51: exemplary embodiments of chimeric co-stimulatory receptors of the present invention. Six CCR constructs using PD-1 or anti-PD-1 (38 A1) scFv extracellular domain (ECD) are shown. TM refers to the transmembrane domain and ICN refers to the intracellular domain.

Fig. 52: pqxix vector backbone diagram, which is an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 53: expression of CCR constructs "CCR4" and "CCR5" (as given in fig. 51) in HEK reporter cells.

Fig. 54: exemplary embodiments of chimeric co-stimulatory receptors of the present invention.

Fig. 55: a domain map comprising the amino acid sequence of 2 CCRs of SP- (38A 1 scFv) - (CD 28 hinge and transmembrane) - (IL-2 Rβ intracellular) -T2A-SP- (19H 9 scFv) - (CD 28 hinge and transmembrane) - (IL-2 Rγ intracellular), using the 38A1 and 19H9 PD-L1 domains described herein (SEQ ID NO: 658).

Fig. 56: a domain map comprising the amino acid sequence of 2 CCRs of SP- (38A 1 scFv) - (CD 28 hinge and transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (19H 9 scFv) - (CD 28 hinge and transmembrane) - (IL-18 RAP intracellular), using the 38A1 and 19H9 PD-L1 domains (SEQ ID NO: 659) described herein.

Fig. 57: a domain map (SEQ ID NO: 660) comprising the amino acid sequence of 2 CCRs of SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2 Rβ transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2 Rγ transmembrane and intracellular).

Fig. 58: a domain map (SEQ ID NO: 661) comprising the amino acid sequence of 2 CCRs of SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18R 1-transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18 RAP-transmembrane and intracellular).

Fig. 59: a domain map (SEQ ID NO: 662) comprising the amino acid sequence of 2 CCRs of SP- (cAR47A6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-2Rβ intracellular) -T2A-SP- (KM 4097 scFv) - (CD 28 hinge and transmembrane) - (IL-2Rγ intracellular).

Fig. 60: a domain map (SEQ ID NO: 663) comprising the amino acid sequence of 2 CCRs of SP- (cAR47A6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (KM 4097scFv scFv) - (CD 28 hinge and transmembrane) - (IL-18 RAP intracellular).

Fig. 61: a pLenti vector diagram, which is an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 62: (A) HEK-IL-18 transduced with the double epitope CCR8 (38A 1scFv-CD28TM-IL-18R1-T2A-19H9scFv-CD28TM-IL-18 RAP) and incubated with biotin conjugated PD-L1 protein reported the results of cells after fluorescent staining with streptavidin; (B) HEK-IL-18 transduced with CCR12 (cAR47A6.4 scFv-CD28TM-IL-18R1-T2A-KM4097scFv-CD28TM-IL-18 RAP) and incubated with biotin conjugated TROP2 protein reported the results of cells after fluorescent staining with streptavidin. Expression of both CCR8 and CCR12 is shown by these results.

Fig. 63: hPD-L1Raji cells were incubated with 38A1-IgG4-HA (hemagglutinin) antibody targeting PD-L1 at the concentrations indicated (FIG. 63) in the presence of the competing hPD-L1 binding antibody 19H 9. After 2 hours of incubation, the cells were washed and stained for anti-HA-APC (carex phycocyanin). The X-axis shows the concentration of titrated 38A1-IgG4-HA antibody and the Y-axis shows the% PD-L1 positive stained cells in total hPD-L1Raji cells.

Fig. 64: hPD-L1Raji cells were incubated with 19H9-IgG4-Flag antibody targeting PD-L1 at the concentrations indicated (FIG. 63) in the presence of competing hPD-L1 binding antibody 38A 1. After 2 hours of incubation, cells were washed and stained for anti-Flag-AF 488. In FIG. 64, the x-axis shows the concentration of titrated 19H9-IgG4-Flag antibody and the Y-axis shows the% PD-L1 positive stained cells in total hPD-L1Raji cells.

Fig. 65: flow cytometry results stained with antibodies shown on each axis.

Fig. 66: effect of AKT inhibitor (AKTi) treatment with two different concentrations of the pan AKT inhibitor epatazobactib (ipaataservib) (0.3 μm and 1 μm) on TIL amplification and viability before REP (pre-REP) and during REP (blue bars) or only during REP phase (purple bars). The amplification and viability of TIL at the end of the 22 day amplification process are shown. Also shown is CD8 of cryopreserved cells after the expansion process ⁺ 、CD4 ⁺ And CD4 ⁺ (Foxp3 ⁺ ) Frequency of cells.

Fig. 67: experimental design to evaluate blocking efficacy of two PD-L1 antibodies (38 A1 and 19H 9).

Fig. 68: experimental results for evaluating blocking efficacy of two PD-L1 antibodies (38 A1 and 19H 9).

Fig. 69: t cell subsets of control and AKT inhibitor (AKTi) -treated TIL. Shows CD8 ⁺ TIL and CD4 ⁺ TIL after treatment T _CM (CD45RA ^- CCR7 ⁺ )、T _EM (CD45RA ^- CCR7 ^- ) And T _EMRA (CD45 ⁺ CCR7 ^- ) Frequency of cells, wherein p is indicated<0.05。

Fig. 70: cytokine and chemokine receptor expression on control and AKT inhibitor (AKTi) -treated TIL. The cryopreserved control or AKTi treated TIL was analyzed by flow cytometry. IL-7R ⁺ And CXCR3 ⁺ CD8 ⁺ Representative histogram of TIL and frequency, p is indicated<0.05 indicates p<0.01。

Fig. 71: CD8 in control and AKT inhibitor (AKTi) treated ⁺ Distribution of CD69 and CD39 single and double positive populations in TIL assessed by flow cytometry, indicative of p<0.05 indicates p<0.01 indicates p<0.001。

Fig. 72: inhibitory receptor and transcription factor on CD69 ^- CD39 ^- And CD69 ⁺ CD39 ⁺ CD8 ⁺ Expression on TIL; PD1, LAG-3, TIM-3 and TIGIT, tbet, eomes, BATF and TOX at CD69 ^- CD39 ^- And CD69 ⁺ CD39 ⁺ Frequency on cells, p is indicated<0.05 indicates p<0.01 indicates p<0.0001. Displayed on CD69 ^- CD39 ^- And CD69 ⁺ CD39 ⁺ CD8 ⁺ Representative histogram and frequency of CD62L expression on TIL.

Fig. 73: control and AKT inhibitor (AKTi) -treated TIL expressed markers after overnight stimulation. Cryopreserved controls and TIL treated with 1 μm AKTi before and at REP at 1:5 against cell scale anti-CD 3/CD28 beads were stimulated overnight. CD69 ^- CD39 ^- And CD69 ⁺ CD39 ⁺ Cell frequency and in CD8 ⁺ Transcription factor expression on TIL, wherein p is indicated<0.05 indicates p<0.01 indicates p<0.001。

Fig. 74: CD8 in control and AKT inhibitor (AKTi) treated ⁺ Cytokine expression on TIL, wherein p is indicated<0.05。

Fig. 75: results of allogeneic cytotoxicity assay. In the left panel, the results show the TIL and the time of the cryopreserved control and AKT inhibitor (epatazobactam) treatment with 1uM both before and during REPTHP-1 cells (Eurofins DiscoverX, friemont, california) at 10:1 effector to target cell ratio co-cultures for 24 hours to measure cytotoxicity in an allogeneic environment. The right panel shows control and AKT inhibitor (AKTi) -treated TIL at 1 every 5 days: results of anti-CD 3/CD28 bead stimulation of the 1 bead versus cell scale. Three days after the third stimulation, cells were washed, beads removed, and cells were washed at 10:1 effector to target cell ratio of KILR THP-1 cells were co-cultured for 24 hours.

Fig. 76: amplification, viability and T cell distribution data of the decitabine treated TIL with increased concentration of control TIL (grey bars) and decitabine (decitabine) are shown. The treatment is added during the REP phase only (blue bars) or before and during the REP phase (green bars). Panel A shows the amplification and viability of TIL at the end of the 22 day amplification process. Panel B shows CD8 of cryopreserved cells analyzed by flow cytometry after the expansion process ⁺ 、CD4 ⁺ And CD4 ⁺ Frequency of (foxp3+) cells. * P (P)<0.05，**P<0.01。

Fig. 77: t cell subsets of control and decitabine treated TILs. TIL T after amplification _CM (CD45RA ^- CCR7 ⁺ )、T _EM (CD45RA ^- CCR7 ^- ) And T _EMRA (CD45 ⁺ CCR7 ^- ) The frequency of cells is shown in panel A (CD 8 ⁺ ) And Panel B (CD 4) ⁺ )。*P<0.05，**P<0.01。

Fig. 78: expression of surface markers on decitabine-treated TIL. The control cryopreserved TIL or decitabine treated cryopreserved TIL was thawed and stained for flow cytometry analysis. Panel A shows that CD25, ICOS, CD28 and IL-7R are on CD8 ⁺ Expression on TIL. Panel B shows that the inhibitory receptors PD-1 and TIGIT are on CD8 ⁺ Expression on TIL. CD4 ⁺ Similar results were observed with TIL. * P (P)<0.05，**P<0.01，***P<0.001，****P<0.0001。

Fig. 79: expression of transcription factors on decitabine-treated TIL. Cryopreservation of control or decitabine treatmentsTIL was thawed and stained for flow cytometry analysis. Shows the Eomes, KLF2, BATF and T-bet at CD8 ⁺ Expression on TIL. * P (P)<0.05，**P<0.01。

Fig. 80: control and decitabine treated TIL expressed cytokines after in vitro stimulation. Cryopreserved control and decitabine treated TIL at 1:5 against cell scale anti-CD 3/CD28 beads were stimulated overnight. Shows IFN-gamma (IFNgamma), TNF-alpha (TNF alpha) and granzyme B (GZMB) at CD8 ⁺ Expression on TIL. * P (P)<0.05，**P<0.01。

Fig. 81: cytotoxicity of control and decitabine treated TIL. In panel A, control TIL was cryopreserved and TIL was treated with 100nM DAC at REP THP-1 cells (Eurofins DiscoverX, friemont, california) at 10:1 effector: target cell ratios were co-cultured for 24h to measure cytotoxicity in an allogeneic environment. In panel B, control and decitabine-treated TIL were stimulated every 5 days with transdcttm (Miltenyi Biotec, germany). One day after the third stimulation, cells were washed at 10:1 effector to target cell ratio KILR THP-1 cells were co-cultured for 24 hours to measure cytotoxicity. * P (P)<0.05。

Fig. 82: control TIL and decitabine-treated TIL were stimulated every 5 days with transdcttm (Miltenyi Biotec, germany). One day after the third stimulation, cells were washed and stained for flow cytometry analysis. Expression of IL-7R, PD-1 and TIM3 of TIL after repeated stimulation is shown in panel A, and expression levels of the transcription factor of TIL after repeated stimulation is shown in panel B. * P <0.05, P <0.01.

Fig. 83: vector design of CCR7.2 bi-epitope CCR targeting PD-L1 using the pLenti framework is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 84: vector design of CCR8.2 bi-epitope CCR targeting PD-L1 using the pLenti framework is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 85: vector design of a CCR11.2 bi-epitope CCR targeting TROP-2 using the pLenti framework is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 86: vector design of CCR12.2 bi-epitope CCR targeting TROP-2 using the slenti framework is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 87: results of HEKIL-18 reporter experiments using CCR (CCR 8 and CCR 8.2) targeting PD-L1 showed that IL-18 signaling was enhanced by using alternative Transmembrane (TM) domains. As described herein, CCR8 is a bi-epitope CCR having the general structure 38A1scFv-CD28TM-IL-18R1-IC-T2A-19H9scFv-CD28TM-IL-18 RAP-IC. CCR8.2 is a double epitope CCR with the general structure 38A1scFv-IL-18R1TM-IL-18R1-IC-T2A-19H9scFV-IL-18RAPTM-IL-18 RAP-IC.

Fig. 88: results of HEKIL-18 reporter experiments using CCR (CCR 12 and CCR 12.2) targeting PD-L1 showed that IL-18 signaling was enhanced by using alternative Transmembrane (TM) domains. As described herein, CCR12 is a bi-epitope CCR having the general structure cad 47a6.4 scFv-CD28TM-IL-18R1-IC-T2A-KM4097scFv-CD28TM-IL-18 RAP-IC. CCR12.2 is a double epitope CCR with the general structure cAR47A6.4scFv-IL-18R 1TM-IL-18R1-IC-T2A-KM4097scFv-IL-18RAPTM-IL-18 RAP-IC.

Fig. 89: the results of the PD-L1 targeting CCR experiments (see FIG. 87) show comparison with different concentrations of IL-18 control.

Fig. 90: the results of TROP-2-targeting CCR experiments (as in fig. 88) show comparison with different concentrations of IL-18 control.

Fig. 91: exemplary CCR designs are primarily for constructs having 4-1BB (CD 137) intracellular domains, which are also embodiments of the present invention. EC means extracellular, TM means transmembrane, SP means signal peptide, and IC means intracellular.

Fig. 92: the use of the pLenti framework to target the CCR13CCR of FAS is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 93: the use of the pLenti framework for the design of a vector targeting CCR14CCR of PD-1 is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 94: the use of the pLenti framework to target the CCR15CCR vector design of tgfbetarii is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 95: vector design of CCR14CCR (with CD28 intracellular domain) targeting PD-1 using the slenti framework is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 96: the CCR construct was inserted into the pLenti-IRES-GFP lentiviral plasmid. TIL was transduced by lentivirus, allowed to stand for 2 days, then amplified using the 11 day REP amplification procedure. The surface expression of the CCR constructs shown here was detected by flow cytometry.

Fig. 97: amplification, viability and killing efficacy of TIL after CCR-expressing REP.

Fig. 98: exemplary CCR designs for constructs with LTBR intracellular domains are also embodiments of the invention. EC means extracellular, TM means transmembrane, SP means signal peptide, and IC means intracellular.

Fig. 99: the use of the pLenti framework to target the CCR17CCR of FAS is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 100: the use of the pLenti framework for the design of a CCR18CCR vector targeting PD-1 is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Fig. 101: the use of the pLenti backbone to target the CCR19CCR of tgfbetarii vector design is also an embodiment of the different CCR and chemokine receptor vectors of the invention.

Description of sequence Listing

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

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

SEQ ID NO:6 is the amino acid sequence of endo-tile Lu Jin alpha.

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

SEQ ID NO:8 is a mucin (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 HCDR2kabat of IgG. IL2R67A. H1.

SEQ ID NO:21 is HCDR3kabat of IgG. IL2R67A. H1.

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

SEQ ID NO:23 is HCDR2clothia of IgG. IL2R67A. H1.

SEQ ID NO:24 is HCDR3clothia of IgG. IL2R67A. H1.

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

SEQ ID NO:26 is HCDR2IMGT of IgG. IL2R67A. H1.

SEQ ID NO:27 is HCDR3IMGT of IgG. IL2R67A. H1.

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

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

SEQ ID NO:30 is LCDR1kabat of IgG. IL2R67A. H1.

SEQ ID NO:31 is LCDR2kabat of IgG. IL2R67A. H1.

SEQ ID NO:32 is LCDR3kabat of IgG. IL2R67A. H1.

SEQ ID NO:33 is LCDR1chothia of IgG. IL2R67A. H1.

SEQ ID NO:34 is LCDR2chothia of IgG. IL2R67A. H1.

SEQ ID NO:35 is LCDR3chothia of IgG. IL2R67A. H1.

SEQ ID NO:36 is V _L A 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 (utomiumab) (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 heavy chain variable region (V) of monoclonal antibody Wu Tumu monoclonal antibody (PF-05082566) 4-1BB agonist _H )。

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

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 monoclonal antibody (PF-05082566).

SEQ ID NO:48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Wu Tumu monoclonal antibody (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 monoclonal antibody (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) of the 4-1BB agonist monoclonal antibody Wu Ruilu mab (BMS-663513) _H )。

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 (V) of 4-1BB agonist antibody 4B4-1-1 version 1 _H )。

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

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

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

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

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

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 Tavolixizumab (MEDI-0562).

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

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

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

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

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

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

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

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

SEQ ID NO:96 is the light chain CDR3 of the OX40 agonist monoclonal antibody Tavliximab (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) of OX40 agonist monoclonal antibody 11D4 _H )。

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

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) of OX40 agonist monoclonal antibody 18D8 _H )。

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

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) of the OX40 agonist monoclonal antibody Hu119-122 _H )。

SEQ ID NO:118 is the light chain variable region (V _L )。

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 )。

SEQ ID NO:126 is the light chain variable region (V _L )。

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 OX40 ligand (OX 40L) amino acid sequence.

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 (V) of OX40 agonist monoclonal antibody 008 _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 )。

SEQ ID NO:139 is the light chain variable region (V _L )。

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

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

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

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

SEQ ID NO:144 is the heavy chain variable region (V _H )。

SEQ ID NO:145 is the light chain variable region (V _L )。

SEQ ID NO:146 is the heavy chain variable region (V _H )。

SEQ ID NO:147 is the light chain variable region (V _L )。

SEQ ID NO:148 is the heavy chain variable region (V _H )。

SEQ ID NO:149 is the heavy chain variable region (V _H )。

SEQ ID NO:150 is the light chain variable region (V _L )。

SEQ ID NO:151 is the light chain variable region (V _L )。

SEQ ID NO:152 is the heavy chain variable region (V _H )。

SEQ ID NO:153 is the heavy chain variable region (V _H )。

SEQ ID NO:154 is the light chain variable region (V _L )。

SEQ ID NO:155 is the light chain variable region (V _L )。

SEQ ID NO:156 is the heavy chain variable region (V _H )。

SEQ ID NO:157 is the light chain variable region (V _L )。

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) of the PD-1 inhibitor nivorunib _H ) Amino acid sequence.

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

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 pembrolizumab.

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

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

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

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

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

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

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

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

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

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

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) of the PD-L1 inhibitor dewaruzumab _H ) Amino acid sequence.

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

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 Evellumab.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 tremelimumab.

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

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

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

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

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

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

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

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

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

SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor Z Li Fu monoclonal antibody.

SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4 inhibitor za Li Fu monoclonal antibody.

SEQ ID NO:230 is the heavy chain variable region of the CTLA-4 inhibitor Z Li Fu monoclonal antibody (V _H ) Amino acid sequence.

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

SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor Z Li Fu monoclonal antibody.

SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor Z Li Fu monoclonal antibody.

SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor Z Li Fu monoclonal antibody.

SEQ ID NO:235 is the light chain CDR1 amino acid sequence of CTLA-4 inhibitor za Li Fu monoclonal antibody.

SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor Z Li Fu monoclonal antibody.

SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor Z Li Fu monoclonal antibody.

SEQ ID NO:238 is the amino acid sequence of the scFv linker.

SEQ ID NO:239 is the amino acid sequence of the scFv linker.

SEQ ID NO:240 is the amino acid sequence of the scFv linker.

SEQ ID NO:241 is the amino acid sequence of the scFv linker.

SEQ ID NO:242 is the amino acid sequence of the scFv linker.

SEQ ID NO:243 is the amino acid sequence of the scFv linker.

SEQ ID NO:244 is the amino acid sequence of the extracellular domain of PD-1.

SEQ ID NO:245 is the amino acid sequence of the extracellular and transmembrane domains of PD-1.

SEQ ID NO:246 is the amino acid sequence of the extracellular domain of PD-1 and the transmembrane domain of CD 28.

SEQ ID NO:247 is the nucleotide sequence of the extracellular and transmembrane domains of PD-1.

SEQ ID NO:248 is the nucleotide sequence of the PD-1 extracellular domain and CD28 transmembrane domain.

SEQ ID NO:249 is the amino acid sequence of scFv-Fc antibody 38 A1.

SEQ ID NO:250 is the amino acid sequence of the variable heavy chain of scFv antibody 38 A1.

SEQ ID NO:251 is the amino acid sequence of the scFv antibody 38A1 variable light chain.

SEQ ID NO:252 is the amino acid sequence of the variable heavy chain CDR1 of scFv antibody 38 A1.

SEQ ID NO:253 is the amino acid sequence of the variable heavy chain CDR2 of scFv antibody 38 A1.

SEQ ID NO:254 is the amino acid sequence of the variable heavy chain CDR3 of scFv antibody 38A 1.

SEQ ID NO:255 is the amino acid sequence of scFv antibody 38A1 variable light chain CDR 1.

SEQ ID NO:256 is the amino acid sequence of scFv antibody 38A1 variable light chain CDR 2.

SEQ ID NO:257 is the amino acid sequence of the CDR3 of scFv antibody 38A1 variable light chain.

SEQ ID NO:258 is the amino acid sequence of scFv-Fc antibody 19H 9.

SEQ ID NO:259 is the amino acid sequence of the scFv antibody 19H9 variable heavy chain.

SEQ ID NO:260 is the amino acid sequence of the scFv antibody 19H9 variable light chain.

SEQ ID NO:261 is the amino acid sequence of the CDR1 of scFv antibody 19H9 variable heavy chain.

SEQ ID NO:262 is the amino acid sequence of the CDR2 of scFv antibody 19H9 variable heavy chain.

SEQ ID NO:263 is the amino acid sequence of CDR3 of scFv antibody 19H9 variable heavy chain.

SEQ ID NO:264 is the amino acid sequence of CDR1 of scFv antibody 19H9 variable light chain.

SEQ ID NO:265 is the amino acid sequence of the CDR2 of scFv antibody 19H9 variable light chain.

SEQ ID NO:266 is the amino acid sequence of CDR3 of scFv antibody 19H9 variable light chain.

SEQ ID NO:267 is an anti-CEA variable heavy chain amino acid sequence.

SEQ ID NO:268 is an anti-CEA variable light chain amino acid sequence.

SEQ ID NO:269 is an anti-CEA heavy chain CDR1 amino acid sequence.

SEQ ID NO:270 is the anti-CEA heavy chain CDR2 amino acid sequence.

SEQ ID NO:271 is the anti-CEA heavy chain CDR3 amino acid sequence.

SEQ ID NO:272 is the anti-CEA light chain CDR1 amino acid sequence.

SEQ ID NO:273 is the anti-CEA light chain CDR2 amino acid sequence.

SEQ ID NO:274 is the anti-CEA light chain CDR3 amino acid sequence.

SEQ ID NO:275 is an anti-CD 73 variable heavy chain amino acid sequence.

SEQ ID NO:276 is an anti-CD 73 variable light chain amino acid sequence.

SEQ ID NO:277 is an anti-CD 73 heavy chain CDR1 amino acid sequence.

SEQ ID NO:278 is an anti-CD 73 heavy chain CDR2 amino acid sequence.

SEQ ID NO:279 is the anti-CD 73 heavy chain CDR3 amino acid sequence.

SEQ ID NO:280 is the anti-CD 73 light chain CDR1 amino acid sequence.

SEQ ID NO:281 is the anti-CD 73 light chain CDR2 amino acid sequence.

SEQ ID NO:282 is the anti-CD 73 light chain CDR3 amino acid sequence.

SEQ ID NO:283 is an anti-CD 73 variable heavy chain amino acid sequence.

SEQ ID NO:284 is an anti-CD 73 variable light chain amino acid sequence.

SEQ ID NO:285 is the anti-CD 73 heavy chain CDR1 amino acid sequence.

SEQ ID NO:286 is the anti-CD 73 heavy chain CDR2 amino acid sequence.

SEQ ID NO:287 is the anti-CD 73 heavy chain CDR3 amino acid sequence.

SEQ ID NO:288 is the anti-CD 73 light chain CDR1 amino acid sequence.

SEQ ID NO:289 is an anti-CD 73 light chain CDR2 amino acid sequence.

SEQ ID NO:290 is the anti-CD 73 light chain CDR3 amino acid sequence.

SEQ ID NO:291 is an anti-TROP-2 variable heavy chain amino acid sequence.

SEQ ID NO:292 is an anti-TROP-2 variable heavy chain amino acid sequence.

SEQ ID NO:293 is an anti-TROP-2 variable heavy chain amino acid sequence.

SEQ ID NO:294 is an anti-TROP-2 variable heavy chain amino acid sequence.

SEQ ID NO:295 is an anti-TROP-2 variable heavy chain amino acid sequence.

SEQ ID NO:296 is an anti-TROP-2 variable heavy chain amino acid sequence.

SEQ ID NO:297 is the anti-TROP-2 variable light chain amino acid sequence.

SEQ ID NO:298 is an anti-TROP-2 variable light chain amino acid sequence.

SEQ ID NO:299 is an anti-TROP-2 variable light chain amino acid sequence.

SEQ ID NO:300 is an anti-TROP-2 variable light chain amino acid sequence.

SEQ ID NO:301 is the anti-TROP-2 heavy chain CDR1 amino acid sequence.

SEQ ID NO:302 is the anti-TROP-2 heavy chain CDR2 amino acid sequence.

SEQ ID NO:303 is the anti-TROP-2 heavy chain CDR3 amino acid sequence.

SEQ ID NO:304 is the anti-TROP-2 light chain CDR1 amino acid sequence.

SEQ ID NO:305 is an anti-TROP-2 light chain CDR2 amino acid sequence.

SEQ ID NO:306 is the anti-TROP-2 light chain CDR3 amino acid sequence.

SEQ ID NO:307 is the amino acid sequence of the variable heavy chain of the anti-TROP-2 antibody m7E 6.

SEQ ID NO:308 is the amino acid sequence of the variable light chain of the anti-TROP-2 antibody m7E 6.

SEQ ID NO:309 is the amino acid sequence of the variable heavy chain of the anti-TROP-2 antibody h7E 6.

SEQ ID NO:310 are the amino acid sequences of the variable light chains of the anti-TROP-2 antibodies m7E6 and h7E6_SVG.

SEQ ID NO:311 is the amino acid sequence of the variable heavy chain of the anti-TROP-2 antibodies h7E6_SVGL and h7E6_SVG.

SEQ ID NO:312 is the amino acid sequence of the variable light chain of the anti-TROP-2 antibody h7E6_SVGL.

SEQ ID NO:313 is the amino acid sequence of the variable heavy chain of the anti-TROP-2 antibody m6G 11.

SEQ ID NO:314 is the amino acid sequence of the variable light chain of the anti-TROP-2 antibody m6G 11.

SEQ ID NO:315 is the amino acid sequence of the variable heavy chain of the anti-TROP-2 antibody h6G 11.

SEQ ID NO:316 is the amino acid sequence of the variable light chain of the anti-TROP-2 antibody h6G 11.

SEQ ID NO:317 is the amino acid sequence of the variable heavy chain of the anti-TROP-2 antibody h6G11-FKG_SF.

SEQ ID NO:318 is the amino acid sequence of the variable light chain of the anti-TROP-2 antibody h6G11-FKG_SF.

SEQ ID NO:319 is the amino acid sequence of the variable heavy chain CDR1 of an anti-TROP-2 antibody.

SEQ ID NO:320 is the amino acid sequence of the variable heavy chain CDR2 of an anti-TROP-2 antibody.

SEQ ID NO:321 is the amino acid sequence of the variable heavy chain CDR3 of an anti-TROP-2 antibody.

SEQ ID NO:322 is the amino acid sequence of the variable light chain CDR1 of an anti-TROP-2 antibody.

SEQ ID NO:323 is the amino acid sequence of the variable light chain CDR2 of an anti-TROP-2 antibody.

SEQ ID NO:324 is the amino acid sequence of the variable light chain CDR3 of an anti-TROP-2 antibody.

SEQ ID NO:325 is a nucleotide sequence encoding the m7E6 variable heavy chain of the anti-TROP-2 antibody.

SEQ ID NO:326 is a nucleotide sequence encoding the m7E6 variable light chain of the anti-TROP-2 antibody.

SEQ ID NO:327 is a nucleotide sequence encoding the variable heavy chain of the anti-TROP-2 antibody h7E 6.

SEQ ID NO:328 is a nucleotide sequence encoding the m7E6 variable light chain of the anti-TROP-2 antibody.

SEQ ID NO:329 is a nucleotide sequence encoding the variable heavy chain of the anti-TROP-2 antibody h7E6_SVGL.

SEQ ID NO:330 is a nucleotide sequence encoding the variable light chain of the anti-TROP-2 antibody h7E6_SVGL.

SEQ ID NO:331 is a nucleotide sequence encoding the variable heavy chain of the anti-TROP-2 antibody m6G 11.

SEQ ID NO:332 is a nucleotide sequence encoding the m6G11 variable light chain of the anti-TROP-2 antibody.

SEQ ID NO:333 is a nucleotide sequence encoding the variable heavy chain of the anti-TROP-2 antibody h6G 11.

SEQ ID NO:334 is a nucleotide sequence encoding the h6G11 variable light chain of the anti-TROP-2 antibody.

SEQ ID NO:335 is a nucleotide sequence encoding the variable heavy chain of the anti-TROP-2 antibody h6G11-FKG_SF.

SEQ ID NO:336 is a nucleotide sequence encoding the variable light chain of the anti-TROP-2 antibody h6G11-FKG-SF.

SEQ ID NO:337 is an anti-TROP-2 Sha Xituo bead mab (sacituzumab) variable heavy chain amino acid sequence.

SEQ ID NO:338 is the anti-TROP-2 Sha Xituo bead mab variable light chain amino acid sequence.

SEQ ID NO:339 is an anti-TROP-2 Sha Xituo bead monoclonal antibody heavy chain CDR1 amino acid sequence.

SEQ ID NO:340 is an anti-TROP-2 Sha Xituo bead monoclonal antibody heavy chain CDR2 amino acid sequence.

SEQ ID NO:341 is the anti-TROP-2 Sha Xituo bead monoclonal antibody heavy chain CDR3 amino acid sequence.

SEQ ID NO:342 is the anti-TROP-2 Sha Xituo bead mab light chain CDR1 amino acid sequence.

SEQ ID NO:343 is the anti-TROP-2 Sha Xituo bead monoclonal antibody light chain CDR2 amino acid sequence.

SEQ ID NO:344 is the anti-TROP-2 Sha Xituo bead monoclonal antibody light chain CDR3 amino acid sequence.

SEQ ID NO:345 is the amino acid sequence of the anti-EPCAM scFv antibody 3-17I scFv.

SEQ ID NO:346 is the amino acid sequence of the anti-EPCAM scFv antibody 7-F17 scFv.

SEQ ID NO:347 is the amino acid sequence of an anti-EPCAM scFv antibody 12-C15 scFv.

SEQ ID NO:348 is the amino acid sequence of the anti-EPCAM scFv antibody 16-G5 scFv.

SEQ ID NO:349 is the amino acid sequence of an anti-EPCAM scFv antibody 17-C20 scFv.

SEQ ID NO:350 is the amino acid sequence of the anti-EPCAM scFv antibody 24-G6 scFv.

SEQ ID NO:351 is the variable heavy chain amino acid sequence of an anti-EPCAM antibody.

SEQ ID NO:352 is the variable light chain amino acid sequence of the anti-EPCAM antibody.

SEQ ID NO:353 is the variable light chain amino acid sequence of the anti-EPCAM antibody.

SEQ ID NO:354 is the variable light chain amino acid sequence of the anti-EPCAM antibody.

SEQ ID NO:355 is the variable light chain amino acid sequence of the anti-EPCAM antibody.

SEQ ID NO:356 is the variable light chain amino acid sequence of the anti-EPCAM antibody.

SEQ ID NO:357 is the variable light chain amino acid sequence of an anti-EPCAM antibody.

SEQ ID NO:358 are anti-EPCAM antibody heavy chain CDR1 amino acid sequences.

SEQ ID NO:359 is the anti-EPCAM antibody heavy chain CDR2 amino acid sequence.

SEQ ID NO:360 is the anti-EPCAM antibody heavy chain CDR3 amino acid sequence.

SEQ ID NO:361 is the anti-EPCAM antibody light chain CDR1 amino acid sequence.

SEQ ID NO:362 is the anti-EPCAM antibody light chain CDR2 amino acid sequence.

SEQ ID NO:363 is the anti-EPCAM antibody light chain CDR3 amino acid sequence.

SEQ ID NO:364 is the nucleotide sequence encoding the anti-EPCAM scFv antibody 3-17I scFv.

SEQ ID NO:365 is a nucleotide sequence encoding an anti-EPCAM scFv antibody 7-F17 scFv.

SEQ ID NO:366 is a nucleotide sequence encoding an anti-EPCAM scFv antibody 12-C15 scFv.

SEQ ID NO:366 is a nucleotide sequence encoding an anti-EPCAM scFv antibody 16-G5 scFv.

SEQ ID NO:367 is a nucleotide sequence encoding an anti-EPCAM scFv antibody 17-C20 scFv.

SEQ ID NO:368 is a nucleotide sequence encoding an anti-EPCAM scFv antibody 24-G6 scFv.

SEQ ID NO:369 is a nucleotide sequence encoding the variable heavy chain of the anti-EPCAM scFv.

SEQ ID NO:370 is a nucleotide sequence encoding an anti-EPCAM scFv variable light chain.

SEQ ID NO:371 is a nucleotide sequence encoding an anti-EPCAM scFv variable light chain.

SEQ ID NO:372 is a nucleotide sequence encoding an anti-EPCAM scFv variable light chain.

SEQ ID NO:373 are nucleotide sequences encoding variable light chains of anti-EPCAM scFv.

SEQ ID NO:374 is a nucleotide sequence encoding an anti-EPCAM scFv variable light chain.

SEQ ID NO:375 is a nucleotide sequence encoding an anti-EPCAM scFv variable light chain.

SEQ ID NO:376 is a nucleotide sequence encoding an anti-EPCAM scFv variable light chain.

SEQ ID NO:377 is the variable heavy chain amino acid sequence of the anti-EPCAM antibody.

SEQ ID NO:378 is an anti-EPCAM antibody variable light chain amino acid sequence.

SEQ ID NO:379 is the anti-EPCAM antibody heavy chain CDR1 amino acid sequence.

SEQ ID NO:380 is the anti-EPCAM antibody heavy chain CDR2 amino acid sequence.

SEQ ID NO:381 is the anti-EPCAM antibody heavy chain CDR3 amino acid sequence.

SEQ ID NO:382 is the anti-EPCAM antibody light chain CDR1 amino acid sequence.

SEQ ID NO:383 is the anti-EPCAM antibody light chain CDR2 amino acid sequence.

SEQ ID NO:384 is the anti-EPCAM antibody light chain CDR3 amino acid sequence.

SEQ ID NO:385 is the amino acid sequence of the variable heavy chain of anti-tissue factor antibody TF 260.

SEQ ID NO:386 is the amino acid sequence of the variable light chain of anti-tissue factor antibody TF 260.

SEQ ID NO:387 is the amino acid sequence of the variable heavy chain of anti-tissue factor antibody TF 196.

SEQ ID NO:388 is the amino acid sequence of the variable light chain of the anti-tissue factor antibody TF 196.

SEQ ID NO:389 is the amino acid sequence of the variable heavy chain of the anti-tissue factor antibody TF 278.

SEQ ID NO:390 is the amino acid sequence of the variable light chain of the anti-tissue factor antibody TF 278.

SEQ ID NO:391 is the amino acid sequence of the variable heavy chain of anti-tissue factor antibody TF 277.

SEQ ID NO:392 is the amino acid sequence of the variable light chain of anti-tissue factor antibody TF 277.

SEQ ID NO:393 is the amino acid sequence of the variable heavy chain of the anti-tissue factor antibody TF 392.

SEQ ID NO:394 is the amino acid sequence of the variable light chain of anti-tissue factor antibody TF 392.

SEQ ID NO:395 is the amino acid sequence of the variable heavy chain of anti-tissue factor antibody TF 9.

SEQ ID NO:396 is the amino acid sequence of the variable light chain of the anti-tissue factor antibody TF 9.

SEQ ID NO:397 is the amino acid sequence of the heavy chain CDR1 of the anti-tissue factor antibody TF 260.

SEQ ID NO:398 is the anti-tissue factor antibody TF260 heavy chain CDR2 amino acid sequence.

SEQ ID NO:399 is the heavy chain CDR3 amino acid sequence of anti-tissue factor antibody TF 260.

SEQ ID NO:400 is the amino acid sequence of the light chain CDR1 of the anti-tissue factor antibody TF 260.

SEQ ID NO:401 is the anti-tissue factor antibody TF260 light chain CDR2 amino acid sequence.

SEQ ID NO:402 is the anti-tissue factor antibody TF260 light chain CDR3 amino acid sequence.

SEQ ID NO:403 is the amino acid sequence of the heavy chain CDR1 of the anti-tissue factor antibody TF 196.

SEQ ID NO:404 is the amino acid sequence of the heavy chain CDR2 of the anti-tissue factor antibody TF 196.

SEQ ID NO:405 is the anti-tissue factor antibody TF196 heavy chain CDR3 amino acid sequence.

SEQ ID NO:406 is the amino acid sequence of the light chain CDR1 of the anti-tissue factor antibody TF 196.

SEQ ID NO:407 is the amino acid sequence of the light chain CDR2 of the anti-tissue factor antibody TF 196.

SEQ ID NO:408 is the anti-tissue factor antibody TF196 light chain CDR3 amino acid sequence.

SEQ ID NO:409 is the amino acid sequence of the heavy chain CDR1 of the anti-tissue factor antibody TF 9.

SEQ ID NO:410 is the anti-tissue factor antibody TF9 heavy chain CDR2 amino acid sequence.

SEQ ID NO:411 is the anti-tissue factor antibody TF9 heavy chain CDR3 amino acid sequence.

SEQ ID NO:412 is the anti-tissue factor antibody TF9 light chain CDR1 amino acid sequence.

SEQ ID NO:413 is the anti-tissue factor antibody TF9 light chain CDR2 amino acid sequence.

SEQ ID NO:414 is the anti-tissue factor antibody TF9 light chain CDR3 amino acid sequence.

SEQ ID NO:415 is the amino acid sequence of the variable heavy chain of an anti-tissue factor antibody.

SEQ ID NO:416 is the amino acid sequence of the variable light chain of the anti-tissue factor antibody.

SEQ ID NO:417 is the amino acid sequence of the variable heavy chain of the anti-tissue factor antibody.

SEQ ID NO:418 is the amino acid sequence of the variable light chain of the anti-tissue factor antibody.

SEQ ID NO:419 is the amino acid sequence of the variable heavy chain of the anti-tissue factor antibody.

SEQ ID NO:420 is the amino acid sequence of the variable light chain of an anti-tissue factor antibody.

SEQ ID NO:421 is the amino acid sequence of the variable heavy chain of an anti-tissue factor antibody.

SEQ ID NO:422 is the amino acid sequence of the variable light chain of the anti-tissue factor antibody.

SEQ ID NO:423 is the amino acid sequence of the variable heavy chain of the anti-tissue factor antibody.

SEQ ID NO:424 is the amino acid sequence of the variable light chain of an anti-tissue factor antibody.

SEQ ID NO:425 is a nucleotide sequence encoding the variable heavy chain of anti-tissue factor antibody TF 260.

SEQ ID NO:426 is a nucleotide sequence encoding the variable light chain of anti-tissue factor antibody TF 260.

SEQ ID NO:427 is a nucleotide sequence encoding the variable heavy chain of anti-tissue factor antibody TF 196.

SEQ ID NO:428 is a nucleotide sequence encoding the variable light chain of anti-tissue factor antibody TF 196.

SEQ ID NO:429 is a nucleotide sequence encoding the variable heavy chain of the anti-tissue factor antibody TF 278.

SEQ ID NO:430 is a nucleotide sequence encoding the variable light chain of the anti-tissue factor antibody TF 278.

SEQ ID NO:431 is a nucleotide sequence encoding the variable heavy chain of anti-tissue factor antibody TF 277.

SEQ ID NO:432 is the nucleotide sequence encoding the variable light chain of anti-tissue factor antibody TF 277.

SEQ ID NO:433 is a nucleotide sequence encoding the variable heavy chain of the anti-tissue factor antibody TF 392.

SEQ ID NO:434 is a nucleotide sequence encoding the variable light chain of the anti-tissue factor antibody TF 392.

SEQ ID NO:435 is a nucleotide sequence encoding the variable heavy chain of anti-tissue factor antibody TF 9.

SEQ ID NO:436 is a nucleotide sequence encoding the variable light chain of the anti-tissue factor antibody TF 9.

SEQ ID NO:437 is the amino acid sequence of the variable heavy chain of an anti-LFA-1 or anti-CD 11a antibody.

SEQ ID NO:438 is the amino acid sequence of the variable light chain of an anti-LFA-1 or anti-CD 11a antibody.

SEQ ID NO:439 is the amino acid sequence of the variable heavy chain of an anti-LFA-1 or anti-CD 11a antibody.

SEQ ID NO:440 is the amino acid sequence of the variable light chain of an anti-LFA-1 or anti-CD 11a antibody.

SEQ ID NO:441 is the anti-LFA-1 or anti-CD 11a antibody heavy chain CDR1 amino acid sequence.

SEQ ID NO:442 is the anti-LFA-1 or anti-CD 11a antibody heavy chain CDR2 amino acid sequence.

SEQ ID NO:443 is the anti-LFA-1 or anti-CD 11a antibody heavy chain CDR3 amino acid sequence.

SEQ ID NO:444 is the amino acid sequence of the light chain CDR1 of an anti-LFA-1 or anti-CD 11a antibody.

SEQ ID NO:445 is the anti-LFA-1 or anti-CD 11a antibody light chain CDR2 amino acid sequence.

SEQ ID NO:446 is the anti-LFA-1 or anti-CD 11a antibody light chain CDR3 amino acid sequence.

SEQ ID NO:447 is the amino acid sequence of the anti-FAP scFv based on sibrotuzumab.

SEQ ID NO:448 is the amino acid sequence of the variable heavy chain of the anti-FAP antibody cetrimide.

SEQ ID NO:449 is the amino acid sequence of the variable light chain of the anti-FAP antibody cetrimide.

SEQ ID NO:450 is the amino acid sequence of the FAP5 variable heavy chain of the anti-FAP antibody.

SEQ ID NO:451 is the amino acid sequence of the FAP5 variable light chain of the anti-FAP antibody.

SEQ ID NO:452 is a nucleotide sequence encoding the variable heavy chain of the anti-FAP antibody sirtuin.

SEQ ID NO:453 is the nucleotide sequence encoding the variable light chain of the anti-FAP antibody cetrimide.

SEQ ID NO:454 is the amino acid sequence of the variable heavy chain of anti-VISTA antibody 1B 8.

SEQ ID NO:455 is the amino acid sequence of the variable light chain of anti-VISTA antibody 1B 8.

SEQ ID NO:456 is the amino acid sequence of the heavy chain CDR1 of anti-VISTA antibody 1B 8.

SEQ ID NO:457 is the amino acid sequence of CDR2 of the heavy chain of anti-VISTA antibody 1B 8.

SEQ ID NO:458 is the amino acid sequence of CDR3 of the heavy chain of anti-VISTA antibody 1B 8.

SEQ ID NO:459 is the amino acid sequence of CDR1 of anti-VISTA antibody 1B8 light chain.

SEQ ID NO:460 is the amino acid sequence of CDR2 of the light chain of anti-VISTA antibody 1B 8.

SEQ ID NO:461 is the amino acid sequence of CDR3 of the light chain of anti-VISTA antibody 1B 8.

SEQ ID NO:462 is the amino acid sequence of the variable heavy chain of anti-VISTA antibody 2C 12.

SEQ ID NO:463 is the amino acid sequence of the variable light chain of anti-VISTA antibody 2C 12.

SEQ ID NO:464 is the amino acid sequence of CDR1 of the anti-VISTA antibody 2C12 heavy chain.

SEQ ID NO:465 is the amino acid sequence of CDR2 of the anti-VISTA antibody 2C12 heavy chain.

SEQ ID NO:466 is the amino acid sequence of CDR3 of the heavy chain of anti-VISTA antibody 2C 12.

SEQ ID NO:467 is the amino acid sequence of CDR1 of the anti-VISTA antibody 2C12 light chain.

SEQ ID NO:468 is the amino acid sequence of CDR2 of the anti-VISTA antibody 2C12 light chain.

SEQ ID NO:469 is the amino acid sequence of CDR3 of the anti-VISTA antibody 2C12 light chain.

SEQ ID NO:470 is the amino acid sequence of the variable heavy chain of the anti-VISTA antibody 1a 12.

SEQ ID NO:471 is the amino acid sequence of the variable light chain of anti-VISTA antibody 1a 12.

SEQ ID NO:472 is the amino acid sequence of CDR1 of the heavy chain of anti-VISTA antibody 1a 12.

SEQ ID NO:473 is the amino acid sequence of the heavy chain CDR2 of anti-VISTA antibody 1A 12.

SEQ ID NO:474 is the amino acid sequence of CDR3 of the heavy chain of anti-VISTA antibody 1a 12.

SEQ ID NO:475 is the amino acid sequence of CDR1 of the light chain of anti-VISTA antibody 1A 12.

SEQ ID NO:476 is the amino acid sequence of CDR2 of the light chain of anti-VISTA antibody 1a 12.

SEQ ID NO:477 is the amino acid sequence of CDR3 of the light chain of anti-VISTA antibody 1a 12.

SEQ ID NO:478 is the amino acid sequence of the 3C5 variable heavy chain of an anti-VISTA antibody.

SEQ ID NO:479 is the amino acid sequence of the variable light chain of anti-VISTA antibody 3C 5.

SEQ ID NO:480 is the amino acid sequence of the CDR1 of the anti-VISTA antibody 3C5 heavy chain.

SEQ ID NO:481 is the amino acid sequence of the CDR2 of the 3C5 heavy chain of an anti-VISTA antibody.

SEQ ID NO:482 is the amino acid sequence of the CDR3 of the anti-VISTA antibody 3C5 heavy chain.

SEQ ID NO:483 is the amino acid sequence of CDR1 of the 3C5 light chain of an anti-VISTA antibody.

SEQ ID NO:484 is the amino acid sequence of CDR2 of the 3C5 light chain of an anti-VISTA antibody.

SEQ ID NO:485 is the amino acid sequence of CDR3 of the 3C5 light chain of an anti-VISTA antibody.

SEQ ID NO:486 is the amino acid sequence of the variable heavy chain of anti-LRRC 15 antibody huM.

SEQ ID NO:487 is the amino acid sequence of the variable light chain of anti-LRRC 15 antibody huM.

SEQ ID NO:488 is the amino acid sequence of the heavy chain CDR1 of anti-LRRC 15 antibody huM.

SEQ ID NO:489 is the amino acid sequence of heavy chain CDR2 of anti-LRRC 15 antibody huM.

SEQ ID NO:490 is the amino acid sequence of the heavy chain CDR3 of anti-LRRC 15 antibody huM.

SEQ ID NO:491 is the amino acid sequence of the light chain CDR1 of anti-LRRC 15 antibody huM.

SEQ ID NO:492 is the amino acid sequence of the light chain CDR2 of anti-LRRC 15 antibody huM.

SEQ ID NO:493 is the amino acid sequence of CDR3 of the light chain of anti-LRRC 15 antibody huM.

SEQ ID NO:494 is the amino acid sequence of the variable heavy chain of the anti-LRRC 15 antibody huad208.4.1.

SEQ ID NO:495 is the amino acid sequence of the variable light chain of the anti-LRRC 15 antibody huad208.4.1.

SEQ ID NO:496 is the amino acid sequence of the anti-LRRC 15 antibody huad208.4.1 heavy chain CDR 1.

SEQ ID NO:497 is the amino acid sequence of the heavy chain CDR2 of the anti-LRRC 15 antibody huad208.4.1.

SEQ ID NO:498 is the amino acid sequence of the heavy chain CDR3 of the anti-LRRC 15 antibody huAD208.4.1.

SEQ ID NO:499 is the amino acid sequence of the anti-LRRC 15 antibody huad208.4.1 light chain CDR 1.

SEQ ID NO:500 is the amino acid sequence of the anti-LRRC 15 antibody huad208.4.1 light chain CDR 2.

SEQ ID NO:501 is the amino acid sequence of the anti-LRRC 15 antibody huad208.4.1 light chain CDR 3.

SEQ ID NO:502 is the amino acid sequence of the variable heavy chain of the anti-LRRC 15 antibody huad208.12.1.

SEQ ID NO:503 is the amino acid sequence of the variable light chain of the anti-LRRC 15 antibody huad208.12.1.

SEQ ID NO:504 is the amino acid sequence of the heavy chain CDR1 of the anti-LRRC 15 antibody huad208.12.1.

SEQ ID NO:505 is the amino acid sequence of the heavy chain CDR2 of the anti-LRRC 15 antibody huad208.12.1.

SEQ ID NO:506 is the amino acid sequence of the heavy chain CDR3 of the anti-LRRC 15 antibody huad208.12.1.

SEQ ID NO:507 is the amino acid sequence of the anti-LRRC 15 antibody huad208.12.1 light chain CDR 1.

SEQ ID NO:508 is the amino acid sequence of the anti-LRRC 15 antibody huad208.12.1 light chain CDR 2.

SEQ ID NO:509 is the amino acid sequence of the anti-LRRC 15 antibody huad208.12.1 light chain CDR 3.

SEQ ID NO:510 is the amino acid sequence of the variable heavy chain of the anti-LRRC 15 antibody huad 208.14.1.

SEQ ID NO:511 is the amino acid sequence of the variable light chain of the anti-LRRC 15 antibody huad208.14.1.

SEQ ID NO:512 is the amino acid sequence of the heavy chain CDR1 of the anti-LRRC 15 antibody huad208.14.1.

SEQ ID NO:513 is the amino acid sequence of the heavy chain CDR2 of the anti-LRRC 15 antibody huad208.14.1.

SEQ ID NO:514 is the amino acid sequence of the heavy chain CDR3 of the anti-LRRC 15 antibody huad208.14.1.

SEQ ID NO:515 is the amino acid sequence of the anti-LRRC 15 antibody huad208.14.1 light chain CDR 1.

SEQ ID NO:516 is the amino acid sequence of the anti-LRRC 15 antibody huad208.14.1 light chain CDR 2.

SEQ ID NO:517 is the amino acid sequence of the anti-LRRC 15 antibody huad208.14.1 light chain CDR 3.

SEQ ID NO:518 is the amino acid sequence of the variable heavy chain of the anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:519 is the amino acid sequence of the variable light chain of the anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:520 is the amino acid sequence of the heavy chain CDR1 of anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:521 is the amino acid sequence of the heavy chain CDR2 of the anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:522 is the amino acid sequence of the heavy chain CDR3 of anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:523 is the amino acid sequence of the light chain CDR1 of the anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:524 is the amino acid sequence of the light chain CDR2 of anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:525 is the amino acid sequence of the light chain CDR3 of anti-LRRC 15 antibody hu 139.10.

SEQ ID NO:526 is the amino acid sequence of the variable heavy chain of the anti-LRRC 15 antibody muad210.40.9.

SEQ ID NO:527 is the amino acid sequence of the variable light chain of the anti-LRRC 15 antibody muad210.40.9.

SEQ ID NO:528 is the amino acid sequence of the heavy chain CDR1 of the anti-LRRC 15 antibody muad 210.40.9.

SEQ ID NO:529 is the amino acid sequence of the anti-LRRC 15 antibody muad210.40.9 heavy chain CDR 2.

SEQ ID NO:530 is the amino acid sequence of the anti-LRRC 15 antibody muad210.40.9 heavy chain CDR 3.

SEQ ID NO:531 is the amino acid sequence of the anti-LRRC 15 antibody muad210.40.9 light chain CDR 1.

SEQ ID NO:532 is the amino acid sequence of the anti-LRRC 15 antibody muad210.40.9 light chain CDR 2.

SEQ ID NO:533 is the amino acid sequence of the anti-LRRC 15 antibody muad210.40.9 light chain CDR 3.

SEQ ID NO:534 is the amino acid sequence of the variable heavy chain of the anti-LRRC 15 antibody muad 209.9.1.

SEQ ID NO:535 is the amino acid sequence of the variable light chain of the anti-LRRC 15 antibody muad 209.9.1.

SEQ ID NO:536 is the amino acid sequence of the heavy chain CDR1 of the anti-LRRC 15 antibody muAD209.9.1.

SEQ ID NO:537 is the amino acid sequence of the heavy chain CDR2 of the anti-LRRC 15 antibody muad 209.9.1.

SEQ ID NO:538 is the amino acid sequence of the heavy chain CDR3 of the anti-LRRC 15 antibody muAD209.9.1.

SEQ ID NO:539 is the amino acid sequence of the light chain CDR1 of the anti-LRRC 15 antibody muAD209.9.1.

SEQ ID NO:540 is the amino acid sequence of the anti-LRRC 15 antibody muad209.9.1 light chain CDR 2.

SEQ ID NO:541 is the amino acid sequence of CDR3 of the light chain of the anti-LRRC 15 antibody muad 209.9.1.

SEQ ID NO:542 is the amino acid sequence of the variable heavy chain of the anti-B7-H3 antibody hBRCA 84D.

SEQ ID NO:543 is the amino acid sequence of the variable light chain of the anti-B7-H3 antibody hBRCA 84D.

SEQ ID NO:544 is the amino acid sequence of CDR1 of the heavy chain of hBRCA84D of the anti-B7-H3 antibody.

SEQ ID NO:545 is the amino acid sequence of CDR2 of the heavy chain of hBRCA84D of the anti-B7-H3 antibody.

SEQ ID NO:546 is the amino acid sequence of CDR3 of the heavy chain of hBRCA84D of the anti-B7-H3 antibody.

SEQ ID NO:547 is the amino acid sequence of CDR1 of the light chain of hBRCA84D of the anti-B7-H3 antibody.

SEQ ID NO:548 is the amino acid sequence of CDR2 of the light chain of the anti-B7-H3 antibody hBRCA 84D.

SEQ ID NO:549 is the amino acid sequence of CDR3 of the light chain of hBRCA84D of the anti-B7-H3 antibody.

SEQ ID NO:550 is the amino acid sequence of the variable heavy chain of the anti-B7-H3 antibody hBRCA 84D.

SEQ ID NO:551 is the amino acid sequence of the variable light chain of the anti-B7-H3 antibody hBRCA 84D.

SEQ ID NO:552 is the amino acid sequence of the PD-1 transmembrane domain.

SEQ ID NO:553 is the amino acid sequence of the CD28 transmembrane domain.

SEQ ID NO:554 is the amino acid sequence of the CD27 transmembrane domain.

SEQ ID NO:555 is the amino acid sequence of the CD8 a transmembrane domain.

SEQ ID NO:556 is the amino acid sequence of the CD8 a hinge domain.

SEQ ID NO:557 is the amino acid sequence of the IL-2Rβ hinge domain.

SEQ ID NO:558 is the amino acid sequence of the IgG1 transmembrane and hinge domain.

SEQ ID NO:559 is the amino acid sequence of an IgG1 hinge domain.

SEQ ID NO:560 is the amino acid sequence of the IgG4 hinge domain.

SEQ ID NO:561 is the amino acid sequence of the IgD hinge domain.

SEQ ID NO:562 is a nucleotide sequence encoding a PD-1 transmembrane domain.

SEQ ID NO:563 is a nucleotide sequence encoding a CD28 transmembrane domain.

SEQ ID NO:564 is a nucleotide sequence encoding a CD27 transmembrane domain.

SEQ ID NO:565 is a nucleotide sequence encoding a CD8 alpha transmembrane domain.

SEQ ID NO:566 is a nucleotide sequence encoding a CD8 alpha hinge domain.

SEQ ID NO:567 is a nucleotide sequence encoding an IL-2rβ hinge domain.

SEQ ID NO:568 are the nucleotide sequences encoding the IgG1 transmembrane and hinge domains.

SEQ ID NO:569 is a nucleotide sequence encoding an IgG1 hinge domain.

SEQ ID NO:570 is the nucleotide sequence encoding the hinge domain of IgG 4.

SEQ ID NO:571 is the nucleotide sequence encoding the hinge domain of IgD.

SEQ ID NO:572 is the amino acid sequence of the intracellular domain of CD 28.

SEQ ID NO:573 is the amino acid sequence of the CD134 (OX 40) intracellular domain.

SEQ ID NO:574 is the amino acid sequence of the intracellular domain of CD278 (ICOS).

SEQ ID NO:575 is the amino acid sequence of the intracellular domain of CD137 (4-1 BB).

SEQ ID NO:576 is the amino acid sequence of the CD27 intracellular domain.

SEQ ID NO:577 is the amino acid sequence of the intracellular domain of CD3 zeta.

SEQ ID NO:578 is the amino acid sequence of the intracellular domain of IL-2R beta.

SEQ ID NO:579 is the amino acid sequence of the intracellular domain of IL-2 Rgamma.

SEQ ID NO:580 is the amino acid sequence of the intracellular domain of IL-18R 1.

SEQ ID NO:581 is the amino acid sequence of the intracellular domain of IL-7Rα.

SEQ ID NO:582 is the amino acid sequence of the intracellular domain of IL-12R 1.

SEQ ID NO:583 is the amino acid sequence of the intracellular domain of IL-12R 2.

SEQ ID NO:584 is the amino acid sequence of the intracellular domain of IL-15Rα.

SEQ ID NO:585 is the amino acid sequence of the intracellular domain of IL-21R.

SEQ ID NO:586 is the amino acid sequence of the intracellular domain of LTBR.

SEQ ID NO:587 is the amino acid sequence of the linker.

SEQ ID NO:588 is a nucleotide sequence encoding the intracellular domain of CD 28.

SEQ ID NO:589 is a nucleotide sequence encoding the intracellular domain of CD134 (OX 40).

SEQ ID NO:590 is a nucleotide sequence encoding the intracellular domain of CD278 (ICOS).

SEQ ID NO:591 is a nucleotide sequence encoding the intracellular domain of CD137 (4-1 BB).

SEQ ID NO:592 is a nucleotide sequence encoding an intracellular domain of CD 27.

SEQ ID NO:593 is a nucleotide sequence encoding the intracellular domain of CD3 ζ.

SEQ ID NO:594 is a nucleotide sequence encoding the intracellular domain of IL-2Rβ.

SEQ ID NO:595 is a nucleotide sequence encoding an intracellular domain of IL-2 Rgamma.

SEQ ID NO:596 is a nucleotide sequence encoding the intracellular domain of IL-18R 1.

SEQ ID NO:597 is a nucleotide sequence encoding an intracellular domain of IL-7Rα.

SEQ ID NO:598 is a nucleotide sequence encoding the intracellular domain of IL-12R 1.

SEQ ID NO:599 is a nucleotide sequence encoding an intracellular domain of IL-12R 2.

SEQ ID NO:600 is a nucleotide sequence encoding an intracellular domain of IL-15Rα.

SEQ ID NO:601 is a nucleotide sequence encoding the intracellular domain of IL-21R.

SEQ ID NO:602 is the nucleotide sequence encoding the intracellular domain of LTBR.

SEQ ID NO:603 is the nucleotide sequence encoding the linker.

SEQ ID NO:604 is the nucleotide sequence of EF-1 promoter.

SEQ ID NO:605 is the nucleotide sequence of the CMV promoter.

SEQ ID NO:606 is the nucleotide sequence of the MSCV promoter.

SEQ ID NO:607 is the nucleotide sequence of the NFAT promoter.

SEQ ID NO:608 is the amino acid sequence of the T2A self-cleaving peptide (derived from the vein occlusion virus 2A).

SEQ ID NO:609 is the amino acid sequence of the P2A self-cleaving peptide (derived from porcine teschovirus-1 2A).

SEQ ID NO:610 is the amino acid sequence of the E2A self-cleaving peptide (derived from equine rhinitis a virus).

SEQ ID NO:611 is the amino acid sequence of the F2A self-cleaving peptide (derived from foot and mouth disease virus).

SEQ ID NO:612 is the amino acid sequence of the linker.

SEQ ID NO:613 is a nucleotide sequence encoding a T2A self-cleaving peptide.

SEQ ID NO:614 is a nucleotide sequence encoding a P2A self-cleaving peptide.

SEQ ID NO:615 is a nucleotide sequence encoding an E2A self-cleaving peptide.

SEQ ID NO:616 is a nucleotide sequence encoding an F2A self-cleaving peptide.

SEQ ID NO:617 is the nucleotide sequence encoding the IRES domain.

SEQ ID NO:618 is code comprising (anti TROP 2-V) _L ) - (linker) - (anti-TROP 2-V) _H ) Nucleotide sequence of the vector for CCR of- (IgG 4 hinge and transmembrane) - (IL-2 Rβ).

SEQ ID NO:619 coding for (anti-FAP-V) _L ) - (linker) - (anti-FAP-V _H ) - (CD 8 alpha hinge)And transmembrane) - (IL-18R 1) of the CCR vector.

SEQ ID NO:620 is a coding sequence comprising (anti-PD-L1-V) _L ) - (linker) - (anti-PD-L1-V) _H ) Nucleotide sequence of the vector for CCR of- (CD 8a hinge and transmembrane) - (IL-2 rβ) using the 38A1 anti-PD-L1 domain described herein.

SEQ ID NO:621 is a nucleotide sequence encoding a vector comprising 2 CCRs of SP- (38A 1 scFv) - (CD 28 hinge and transmembrane) - (IL-2 Rβintracellular) -T2A-SP- (19H 9 scFv) - (CD 28 hinge and transmembrane) - (IL-2 Rγintracellular), using the 38A1 and 19H9 PD-L1 domains described herein. SP refers to a signal peptide.

SEQ ID NO:622 is a nucleotide sequence encoding a vector comprising 2 CCRs of SP- (38A 1 scFv) - (CD 28 hinge and transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (19H 9 scFv) - (CD 28 hinge and transmembrane) - (IL-18 RAP intracellular), using the 38A1 and 19H9 PD-L1 domains described herein. SP refers to a signal peptide.

SEQ ID NO:623 is a nucleotide sequence encoding a vector comprising 2 CCRs of SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2 Rβ transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2 Rγ transmembrane and intracellular). SP refers to a signal peptide.

SEQ ID NO:624 is a nucleotide sequence encoding a vector comprising 2 CCR of SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18R 1-transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18 RAP-transmembrane and intracellular). SP refers to a signal peptide.

SEQ ID NO:625 is a nucleotide sequence encoding a vector comprising 2 CCRs of SP- (cAR47A6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-2Rβ intracellular) -T2A-SP- (KM 4097 scFv) - (CD 28 hinge and transmembrane) - (IL-2Rγ intracellular). SP refers to a signal peptide.

SEQ ID NO:626 is a nucleotide sequence encoding a vector comprising 2 CCRs of SP- (cAR47A6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (KM 4097 scFv) - (CD 28 hinge-transmembrane) - (IL-18 RAP intracellular). SP refers to a signal peptide.

SEQ ID NO:627 is the amino acid sequence of the CXCR1 domain.

SEQ ID NO:628 is the amino acid sequence of the 1 and 2 domains of CXCR2 variants.

SEQ ID NO:629 is the amino acid sequence of the CXCR3 variant 1 domain.

SEQ ID NO:630 is the amino acid sequence of the CXCR3 variant 2 domain.

SEQ ID NO:631 is the amino acid sequence of the CXCR4 variant 1 domain.

SEQ ID NO:632 is the amino acid sequence of the CXCR4 variant 2 domain.

SEQ ID NO:633 is the amino acid sequence of the CXCR4 variant 3 domain.

SEQ ID NO:634 is the amino acid sequence of the CXCR4 variant 4 domain.

SEQ ID NO:635 is the amino acid sequence of the CXCR4 variant 5 domain.

SEQ ID NO:636 is the amino acid sequence of the CXCR5 variant 1 domain.

SEQ ID NO:637 is the amino acid sequence of the CXCR5 variant 2 domain.

SEQ ID NO:638 is the amino acid sequence of the a domain of the CCR2 variant.

SEQ ID NO:639 is the amino acid sequence of the B domain of CCR2 variant.

SEQ ID NO:640 is the amino acid sequence of the CCR4 domain.

SEQ ID NO:641 is the amino acid sequence of domains 1 and 2 of the CCR6 variant.

SEQ ID NO:642 is the amino acid sequence of the CCR7 variant 1 domain.

SEQ ID NO:643 is the amino acid sequence of the CCR7 variant 2 domain.

SEQ ID NO:644 is the amino acid sequence of domains 3, 4 and 5 of CCR7 variants.

SEQ ID NO:645 is the amino acid sequence of the CCR8 domain.

SEQ ID NO:646 is a nucleotide sequence encoding a CXCR1 domain.

SEQ ID NO:647 is a nucleotide sequence encoding a CXCR2 variant 1 domain.

SEQ ID NO:648 is a nucleotide sequence encoding a CXCR2 variant 2 domain.

SEQ ID NO:649 is a nucleotide sequence encoding a CXCR3 variant 1 domain.

SEQ ID NO:650 is a nucleotide sequence encoding a CXCR3 variant 2 domain.

SEQ ID NO:651 is a nucleotide sequence encoding a CXCR4 variant 1 domain.

SEQ ID NO:652 is a nucleotide sequence encoding a CXCR4 variant 2 domain.

SEQ ID NO:653 is a nucleotide sequence encoding a CXCR4 variant 3 domain.

SEQ ID NO:654 is a nucleotide sequence encoding a CXCR4 variant 4 domain.

SEQ ID NO:655 is a nucleotide sequence encoding a CXCR4 variant 5 domain.

SEQ ID NO:656 is a nucleotide sequence encoding a CXCR5 variant 1 domain.

SEQ ID NO:657 are nucleotide sequences encoding CXCR5 variant 2 domains.

SEQ ID NO:658 is the nucleotide sequence encoding the A domain of the CCR2 variant.

SEQ ID NO:659 are nucleotide sequences encoding the B domain of CCR2 variants.

SEQ ID NO:660 is a nucleotide sequence encoding a CCR4 domain.

SEQ ID NO:661 is a nucleotide sequence encoding a CCR6 variant 1 domain.

SEQ ID NO:662 is a nucleotide sequence encoding a CCR6 variant 2 domain.

SEQ ID NO:663 is a nucleotide sequence encoding the CCR7 variant 1 domain.

SEQ ID NO:664 is a nucleotide sequence encoding a CCR7 variant 2 domain.

SEQ ID NO:665 is a nucleotide sequence encoding the 3 domain of the CCR7 variant.

SEQ ID NO:666 is a nucleotide sequence encoding the CCR7 variant 4 domain.

SEQ ID NO:667 is a nucleotide sequence encoding the CCR7 variant 5 domain.

SEQ ID NO:668 is a nucleotide sequence encoding a CCR8 domain.

SEQ ID NO:669 are nucleotide sequences encoding vectors for CXCR1 chemokine receptors.

SEQ ID NO:670 is the nucleotide sequence of the vector encoding the CCR8 chemokine receptor.

SEQ ID NO:671 is an amino acid sequence of 2 CCR comprising SP- (38 A1 scFv) - (CD 28 hinge and transmembrane) - (IL-2 rβ intracellular) -T2A-SP- (19 h9 scFv) - (CD 28 hinge and transmembrane) - (IL-2 rγ intracellular) using the 38A1 and 19h9 PD-L1 domains described herein. SP refers to a signal peptide.

SEQ ID NO:672 is the amino acid sequence of 2 CCRs comprising SP- (38A 1 scFv) - (CD 28 hinge and transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (19H 9 scFv) - (CD 28 hinge and transmembrane) - (IL-18 RAP intracellular), using the 38A1 and 19H9 PD-L1 domains described herein. SP refers to a signal peptide.

SEQ ID NO:673 is the amino acid sequence of 2 CCRs comprising SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2 Rβ transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2 Rγ transmembrane and intracellular). SP refers to a signal peptide.

SEQ ID NO:674 is the amino acid sequence of 2 CCRs comprising SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18R 1-transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18 RAP-transmembrane and intracellular). SP refers to a signal peptide.

SEQ ID NO:675 is the amino acid sequence of 2 CCRs comprising SP- (cAR47A6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-2Rβ intracellular) -T2A-SP- (KM 4097 scFv) - (CD 28 hinge and transmembrane) - (IL-2Rγ intracellular). SP refers to a signal peptide.

SEQ ID NO:676 is the amino acid sequence of 2 CCRs comprising SP- (cAR47A6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (KM 4097 scFv) - (CD 28 hinge-transmembrane) - (IL-18 RAP intracellular). SP refers to a signal peptide.

SEQ ID NO:677 is a polypeptide comprising CCR7.2: the amino acid sequence of 2 CCRs of chPD-L1-IL-2R (SP-38A 1scFv-IL2 Rbeta 12aaEC-TM-IL-2 Rbeta-IC-T2A-SP-19H 9scFv-IL2 Rgamma 12aaEC-TM-IL-2 Rgamma-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

SEQ ID NO:678 is a polypeptide comprising CCR8.2: the amino acid sequence of 2 CCRs of chPD-L1-IL-18R (SP-38A 1scFv-IL-18R112aaEC-TM-IL-18R1-IC-T2A-SP-19H9scFv-IL-18RRAP12aaEC-TM-IL-18 RAP-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

SEQ ID NO:679 is a polypeptide comprising CCR11.2: amino acid sequence of 2 CCR of TROP2-IL-2R (SP-cAR47A6.4 scFv-IL2 Rbeta 12aaEC-TM-IL-2 Rbeta-IC-T2A-SP-KM 4097scFV-IL2 Rgamma 12aaEC-TM-IL-2 Rgamma-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

SEQ ID NO:680 is a polypeptide comprising CCR12.2: amino acid sequence of 2 CCR of TROP2-IL-18R (SP-cAR47A6.4 scFv-IL-18R112aaEC-TM-IL-18R1-IC-T2A-SP-KM4097scFv-IL-18RRAP12aaEC-TM-IL-18 RAP-IC). SP refers to signal peptide, EC refers to extracellular, TM refers to transmembrane, and IC refers to intracellular.

SEQ ID NO:681 is the nucleotide sequence encoding CCR 7.2.

SEQ ID NO:682 is a nucleotide sequence encoding CCR 8.2.

SEQ ID NO:683 is the nucleotide sequence encoding CCR 11.2.

SEQ ID NO:684 is the nucleotide sequence encoding CCR 12.2.

SEQ ID NO:685 is the nucleotide sequence of the vector encoding CCR 7.2.

SEQ ID NO:686 is the nucleotide sequence of the vector encoding CCR 8.2.

SEQ ID NO:687 is the nucleotide sequence of the vector encoding CCR 11.2.

SEQ ID NO:688 is the nucleotide sequence of the vector encoding CCR 12.2.

SEQ ID NO:689 is the amino acid sequence of CCR13 (chFas-4-1 BB).

SEQ ID NO:690 is the amino acid sequence of CCR14 (ch PD-1-4-1 BB).

SEQ ID NO:691 is the amino acid sequence of CCR15 (TGF beta RII-4-1 BB).

SEQ ID NO:692 is the amino acid sequence of CCR16 (ch PD-1-CD 28).

SEQ ID NO:693 is the amino acid sequence of the FAS binding domain.

SEQ ID NO:694 is the amino acid sequence of the tgfbetarii binding domain.

SEQ ID NO:695 is the nucleotide sequence encoding CCR13 (chFas-4-1 BB).

SEQ ID NO:696 is the nucleotide sequence encoding CCR14 (chPD-1-4-1 BB).

SEQ ID NO:697 is the nucleotide sequence encoding CCR15 (TGF beta RII-4-1 BB).

SEQ ID NO:698 is the nucleotide sequence encoding CCR16 (chPD-1-CD 28).

SEQ ID NO:699 is the nucleotide sequence of the vector encoding CCR13 (chFas-4-1 BB).

SEQ ID NO:700 is the nucleotide sequence of the vector encoding CCR14 (chPD-1-4-1 BB).

SEQ ID NO:701 is the nucleotide sequence of the vector encoding CCR15 (chTGFβRII-4-1 BB).

SEQ ID NO:702 is the nucleotide sequence of the vector encoding CCR16 (chPD-1-CD 28).

SEQ ID NO:703 is the amino acid sequence of CCR17 (chFas-LTBR).

SEQ ID NO:704 is the amino acid sequence of CCR18 (chPD-1-LTBR).

SEQ ID NO:705 is the amino acid sequence of CCR19 (ch TGF beta RII-LTBR).

SEQ ID NO:706 is a nucleotide sequence encoding CCR17 (chFas-LTBR).

SEQ ID NO:707 is a nucleotide sequence encoding CCR18 (chPD-1-LTBR).

SEQ ID NO:708 is the nucleotide sequence encoding CCR19 (chTGFβRII-LTBR).

SEQ ID NO:709 is the nucleotide sequence of the vector encoding CCR17 (chFas-LTBR).

SEQ ID NO:710 is the nucleotide sequence of the vector encoding CCR18 (chPD-1-LTBR).

SEQ ID NO:711 is the nucleotide sequence of the vector encoding CCR19 (chTGFβRII-LTBR).

SEQ ID NO:712 is the amino acid sequence of CCR20 (ch 19H9-4-1 BB).

SEQ ID NO:713 is the amino acid sequence of CCR21 (ch 19H 9-LTBR).

SEQ ID NO:714 is the amino acid sequence of CCR22 (ch 19H9-4-1BB version 2).

SEQ ID NO:715 is the amino acid sequence of CCR23 (ch 19H9-LTBR version 2).

SEQ ID NO:716 is the amino acid sequence of CCR24 (ch 19H9-LTBR-4-1 BB).

SEQ ID NO:717 is the amino acid sequence of CCR25 (ch 19H9-4-1 BB-LTBR).

SEQ ID NO:718 is the nucleotide sequence encoding CCR20 (ch 19H9-4-1 BB).

SEQ ID NO:719 is a nucleotide sequence encoding CCR21 (ch 19H 9-LTBR).

SEQ ID NO:720 is the nucleotide sequence encoding CCR22 (ch 19H9-4-1BB version 2).

SEQ ID NO:721 is the nucleotide sequence encoding CCR23 (ch 19H9-LTBR version 2).

SEQ ID NO:722 is a nucleotide sequence encoding CCR24 (ch 19H9-LTBR-4-1 BB).

SEQ ID NO:723 is a nucleotide sequence encoding CCR25 (ch 19H9-4-1 BB-LTBR).

Detailed Description

I. Introduction to the invention

Adoptive cell therapy with TIL cultured ex vivo using the Rapid Expansion Protocol (REP) has resulted in successful post-host immunosuppression adoptive cell therapy in cancer (e.g., melanoma) patients. Current TIL manufacturing and therapeutic procedures are limited by length, cost, sterility considerations, and other factors described herein. There is an urgent need to provide a TIL manufacturing process and therapies based on that process (applicable to treat patients with little or no viable treatment options). The present invention meets this need by providing manufacturing processes and products for producing TIL that are modified using CCR or other modifications described herein, etc., to improve their efficacy, potency, safety, dryness, or other efficacy metrics.

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.

The terms "co-administration," "administration in combination with … …," "simultaneously," and "concurrently" as used herein encompass administration of two or more active pharmaceutical ingredients (in preferred embodiments 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 of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Preferably, the separate compositions are administered simultaneously with the administration of the composition in which both agents are present.

The term "in vivo" refers to an event that occurs within the body of 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 that employ living or dead cells, and also cell-free assays that do not employ intact cells.

The term "ex vivo" refers to an event that involves a treatment or procedure performed on cells, tissues and/or organs 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 antigen-specific TIL by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a period of 1 week in number, preferably at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a period of 1 week, or most preferably at least about 100-fold over a period of 1 week. Some rapid amplification protocols are described herein.

"tumor infiltrating lymphocytes" or "TILs" herein refer to a population of cells that are originally obtained as white blood cells but which leave the subject's blood stream and migrate into the tumor. TIL includes but is not limited to CD8 ⁺ Cytotoxic T cells (lymphocytes), th1 and Th17CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TIL includes primary and secondary TIL. "Primary TIL" are those cells obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly collected"), while "secondary TIL" is any population of expanded or proliferated TIL cells 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.

"cell population" (including TIL) as used hereinRefers to some cells that have a common trait. Generally, populations are typically between 1×10 in number ⁶ Up to 1X 10 ¹⁰ Different TIL populations contain different numbers. For example, initial growth of primary TIL in the presence of IL-2 results in about 1X 10 ⁸ A host TIL population of individual cells. REP amplification is usually performed to provide 1.5X10 ⁹ Up to 1.5X10 ¹⁰ A population of cells for infusion.

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

"thawed cryopreserved TIL" as used herein refers to a population of TILs that were previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures where TILs may be administered to patients.

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

The term "cryopreservation medium" refers to any medium that can be used to cryopreserve cells. The medium may comprise a medium comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, hyperthermasol, and combinations thereof. The term "CS10" refers to a cryopreservation medium obtained from Stemcell Technologies or Biolife Solutions. CS10 Medium can be given its trade name'CS10 "is referred to. The CS10 culture medium is a culture medium containing DMSO and containing no serum and animal components 。

The term "central memory T cell" refers to a subset of T cells that are cd45r0+ in humans and constitutively express CCR7 (CCR 7) ^hi ) And CD62L (CD 62) ^hi ). 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 dominant in CD4 cells in blood and are moderately enriched in humans in lymph nodes and tonsils.

The term "effector memory T cells" refers to a subset of human or mammalian T cells, for example, whose central memory T cells are CD45R0+, but have lost the ability to constitutively express CCR7 (CCR 7) ^lo ) And the ability to express CD62L is heterogeneous or low (CD 62L) ^lo ). The surface phenotype of the central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high amounts of inflammatory cytokines including interferon gamma, IL-4 and IL-5 following antigen stimulation. Effector memory T cells are dominant in CD8 cells in blood and are proportionally enriched in the lungs, liver and gut in humans. Cd8+ effector memory T cells carry large amounts of perforin.

The term "closed system" refers to a system that is isolated from 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 is not limited to, a containment G-vessel. Once the tumor segment is added to the closed system, the system is isolated from the external environment until the TIL is ready for administration to the patient.

The terms "morcellation", "fragmentation" and "disruption" as used herein describe the process of destroying a tumor, including mechanical morcellation methods such as breaking, incising, sectioning and fragmenting 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 (PBMC is an antigen presenting cell), 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 PBL is isolated from whole blood or blood cell separation products of the donor. In some embodiments, the PBLs are isolated by positively or negatively selecting a T cell phenotype (e.g., a cd3+cd45+ T cell phenotype) to isolate the product from whole blood or blood cells of the donor.

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, including human, humanized, chimeric or murine antibodies. anti-CD 3 antibodies include OKT-3, also known as molumab. anti-CD 3 antibodies also include UHCT1 clones, also known as T3 and CD3 epsilon. Other anti-CD 3 antibodies include, for example, oxyzumab (otelizumab), tellizumab (teplizumab), 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, and includes commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD pure, miltenyi Biotech, inc., san diego, california) and moromiab or a variant, conservative amino acid substitution, glycosylated form, or biological analogue 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 hybridomas producing OKT-3 were deposited at American type culture Collection (American Type Culture Collection) and assigned ATCC accession No. CRL 8001. The hybridomas producing OKT-3 are also registered in the European authentication cell culture center (ECACC) assigned catalog 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, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biological analogs and variants thereof. IL-2 is described, for example, in Nelson, J.Immunol.2004,172,3983-88 and Malek, annu.Rev.Immunol.2008,26,453-79, the disclosures of which are incorporated herein by reference in their entirety. The amino acid sequences of recombinant human IL-2 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available from multiple suppliers, 22 million IU per single use vial) and recombinant forms of IL-2 supplied by CellGenix, inc., portsmouth, NH, USA (CELLGRO GMP) or Prospec-Tany Technoge Ltd. (Doberoren Rake, N.J.), product number CYT-209-b, as well as other commercial equivalents from other suppliers. Albumin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of about 15kDa. 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 Jiade Lu Jin (NKTR-214, shown as SEQ ID NO:4 PEGylated human recombinant IL-2 wherein an average of 6 lysine residues are trans [ (2, 7-bis { [ methyl poly (oxyethylene) groups) ]Carbamoyl } -9H-fluoren-9-yl) methoxy]Carbonyl-substituted N ⁶ ) Which may be obtained from Nektar Therapeutics (san francisco, california) or may be prepared by methods known in the art, such as the methods described in example 19 of international patent application publication No. WO 2018/132496 A1 or the methods described in example 1 of U.S. patent application publication No. US 2019/0275133 A1, the disclosures of which are incorporated herein by reference in their entirety. Bei Jiade Lu Jin (NKTR-214) and other pegylated IL-2 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 in their entirety. 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 4902502,the disclosure of which is incorporated herein by reference in its entirety. 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 its entirety.

In some embodiments, IL-2 suitable for use in the present invention is in the form THOR-707 available from Synthox, inc. The preparation and nature of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. patent application publication Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosures of which are incorporated herein by reference in their entirety. 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 a conjugate moiety that binds to 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 of the isolated and purified IL-2 polypeptide, 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 position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, the amino acid residue selected from the group consisting of K35, 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 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, 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 of the present invention, in some embodiments, unnatural amino acids include N6-azidoethoxy-L-lysine (AzK), N6-propynylethoxy-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-propargyloxyphenyl alanine, 3-methyl-phenylalanine, levodopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl, O-methyl-L-allyl, O-4-tyrosine, p-serine, p-acetyl-L-tyrosine, 3-phosphono-tyrosine, p-serine, p-phosphono-tyrosine, p-tyrosine, 2-amino-3- ((2- ((3- (benzyloxy) -3-oxopropyl) amino) ethyl) seleno-alkyl (selanyl)) propionic acid, 2-amino-3- (phenylseleno-alkyl) propionic acid, or selenocysteine. In some embodiments, the IL-2 conjugate has a reduced affinity for IL-2 receptor alpha (IL-2Ralpha) subunits relative to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is a reduction in binding affinity for IL-2rα by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater than 99% relative to a 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 than 1000-fold relative to the wild-type IL-2 polypeptide. In some embodiments, the conjugate moiety compromises 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, poly (oxyethylated polyol), poly (enol), poly (vinylpyrrolidone), poly (hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly (saccharide), poly (α -hydroxy acid), poly (vinyl alcohol), polyphosphazene, polyoxazoline (POZ), 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 dextran, 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, or transthyretin. In some embodiments, the proteins each independently comprise an Fc portion. In some embodiments, the proteins each independently comprise an Fc portion of 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 glutamyl. 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, the homobifunctional linker comprises the lomat reagent dithiobis (succinimidyl propionate) (DSP), 3' -dithiobis (sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfodst), ethylene sugar bis (succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N, N ' -disuccinimidyl carbonate (DSC), hexamethylenedime (DMA), heptanediiminedimethyl (DMP), octanediimidyl (DMS), dimethyl-3, 3' -Dithiodipropionamide (DTBP), 1, 4-bis- (3 ' - (2 ' -pyridyldithio) propionylamino) butane (DPDCB), bis-maleimidohexane (BMH), aryl-containing halide compounds (DFDNB) such as 1, 5-difluoro-2, 4-dinitrobenzene or 1, 3-difluoro-4, 6-dinitrobenzene, 4' -difluoro-3, 3' -dinitrophenyl sulfone (DFDNPS), bis- [ beta- (4-azidosalicylamino) ethyl ] disulfide (BASED), formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3 '-dimethylbenzidine, benzidine, α' -p-diaminodiphenyl, diiodo-p-xylene sulphonic 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), long chain N-succinimidyl 3- (2-pyridyldithio) propionate (LC-sPDP), water soluble long chain N-succinimidyl 3- (2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene (sMPT), sulfosuccinimidyl-6- [ α -methyl- α - (2-pyridyldithio) toluylamino ] hexanoate (sulfo-LC-sMPT), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzyloxy-N-hydroxysuccinimide (MBs), bis-succinimidyl-N-hydroxysuccinimide (MBs), and succinimidyl-4- (N-succinimidyl) benzoate (mbb), sulfonyl succinimide (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-maleimidobutyloxy) sulfosuccinimidyl (sulfo-GMBs), succinimidyl 6- ((iodoacetyl) amino) hexanoate (sIAX), succinimidyl 6- [6- (((iodoacetyl) amino ] hexanoate (slAXX), succinimidyl 4- (((iodoacetyl) amino) methyl) cyclohexane-1-carboxylate (sC), succinimidyl 6- (((((4-iodoacetyl) amino) methyl) cyclohexane-1-carbonyl) amino) hexanoate (sIAC), succinimidyl 6- ((iodoacetyl) amino) hexanoate (sIAX), succinimidyl 6- [6- (((iodoacetyl) amino ] hexanoate (sAXX), succinimidyl-4- (p-phenylhydrazine) cross-linking agents such as N- (4-phenylsulfonyl) cross-linking agents, 4- (N-maleimidomethyl) cyclohexane-1-carboxy-hydrazide-8 (M2C 2H), 3- (2-pyridyldithio) propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl- (4-azidosalicylamino) hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2- (p-azidosalicylamino) ethyl-1, 3' -dithiopropionate (sASD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-hydroxysuccinimidyl-6- (4 ' -azido-2 ' -nitrophenylamino) hexanoate (sAH), sulfosuccinimidyl-6- (4 ' -azido-2 ' -azido) nitro-hexanoate (NPAH), N-nitro-5-azido-phenylmethyl-2-nitro-hexanoate (NPAH), sulfosuccinimide-2- (m-azido-nitrobenzoylamino) -ethyl-1, 3' -dithiopropionate (sAND), N-succinimidyl-4 (4-azidophenyl) 1,3' -dithiopropionate (sADP), N-Succinimide (4-azidophenyl) -1,3' -dithiopropionate (sulfo-sADP), sulfosuccinimide 4- (p-azidophenyl) butyrate (sulfo-sAPB), sulfosuccinimide 2- (7-azido-4-methylcoumarin-3-acetamide) ethyl-1, 3' -dithiopropionate (sAED), sulfosuccinimide 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenylazido pyruvate (pNPDP), p-nitrophenyl-2-diazon-3, 3-trifluoropropionate (DTP), 1- (p-salicylamino) -4- (iodoacetamido) butane (AsIB), N- [4- (p-azidophenyl) ethyl-1, 3' -dithiopropionate (PNP), sulfosuccinimide 7-azido-4-methylcoumarin-3-methyl-acetate (sulfo-sAND), p-nitrophenyl-azido-pyruvic acid ester (pNPDP), 4- (p-azidosalicylamino) 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 p-aminobenzyl alcohol (PAB), p-aminooxycarbonyl (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 form of IL-2 suitable for use in the present invention is a fragment of any of the forms of IL-2 described herein. In some embodiments, the form of IL-2 suitable for use in the present invention is pegylation as disclosed in U.S. patent application publication No. US 2020/0181220 A1 and U.S. patent application publication No. US 2020/0330601 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-L-lysine (AzK) covalently attached 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; azK substitution reference SEQ ID NO:5, amino acid positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72. In some embodiments, the IL-2 polypeptide is represented by SEQ ID NO:5 comprises an N-terminal residue. In some embodiments, the form of IL-2 suitable for use in the present invention lacks IL-2Rα chain engagement but retains normal binding 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-L-lysine (AzK) covalently attached 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; azK substitution reference SEQ ID NO:5, amino acid 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 an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached 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; azK substitution reference SEQ ID NO:5, amino acid 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 an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached 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; azK substitution reference SEQ ID NO:5, amino acid 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 inner tile Lu Jin alpha (also known as ALKS-4230 (SEQ ID NO: 6)), which is available from Alkermes, inc. Inner tile Lu Jin alpha is also known as human interleukin 2 fragment (1-59) variant (Cys ¹²⁵ >Ser ⁵¹ ) Through peptide-based linker [ ] ⁶⁰ GG ⁶¹ ) Fused to human interleukin-2 fragment (62-132) via a peptidyl linker @ ¹³³ GSGGGS ¹³⁸ ) Fused to a human interleukin 2 receptor alpha chain fragment (139-303), produced in Chinese Hamster Ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133) -peptide [ Cys ] ¹²⁵ (51)>Ser]Mutant (1-59), by G ₂ Peptide linker (60-61) was fused to human interleukin 2 (IL-2) (4-74) -peptide (62-132) and passed through GSG ₃ S peptide linker (133-138) fused to human interleukin 2 receptor alpha chain (IL 2R subunit alpha, IL2 Ralpha, IL2 RA) (1-165) -peptide (139-303)Glycosylated form alpha is produced in Chinese Hamster Ovary (CHO) cells. The amino acid sequence of endo tile Lu Jin alpha is set forth in SEQ ID NO: 6. In some embodiments, endo tile Lu Jin alpha exhibits the following post-translational modifications: disulfide bonds at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (numbering using SEQ ID NO: 6), and glycosylation sites at the following positions: n187, N206, T212 (numbering using SEQ ID NO: 6). The preparation and nature of inner tile Lu Jin alpha and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. patent application publication No. US 2021/0038684 A1 and U.S. patent No. 10,183,979, the disclosures of which are incorporated herein by reference in their entirety. 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 fusion protein comprising the amino acid sequence of SEQ ID NO:7 or a variant, fragment or derivative thereof. In some embodiments, the form of IL-2 suitable for use in the present invention is a fusion protein comprising a sequence that hybridizes to SEQ ID NO:7 or a variant, fragment or derivative thereof having 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 in its entirety. 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 has receptor antagonist activity of IL-rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, the mucin domain polypeptide linker comprising the amino acid sequence of SEQ ID NO:8 or with SEQ ID NO:8 having at least 90% sequence identity compared to the first fusion partner Fusion of a chaperone with a second fusion partner in the absence of a mucin domain polypeptide linker, the half-life of the fusion protein is improved.

Table 2: amino acid sequence of interleukin

In some embodiments, IL-2 forms suitable for use in the present invention include antibody cytokine implants proteins comprising a heavy chain variable region (V _H ) Light chain variable region (V) _L ) And implant into V _H Or V _L The heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, HCDR3, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, or a fragment thereof, wherein the antibody cytokine implant protein preferentially amplifies T effector cells over regulatory T cells. In one embodiment, the antibody cytokine implant protein comprises a heavy chain variable region (V _H ) Light chain variable region (V) _L ) And implant into V _H Or V _L The heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, HCDR3, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, or a fragment thereof, wherein the IL-2 molecule is a mutein and the antibody cytokine implantation protein preferentially amplifies T effector cells over regulatory T cells. In one embodiment, the IL-2 regimen comprises administration of an antibody as described in U.S. patent application publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the antibody cytokine implant protein comprises a heavy chain variable region (VH), a light chain variable region (VL), and an implant into V _H Or V _L An IL-2 molecule or fragment thereof in the CDRs of (1), the heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2, HCDR3, the light chain variable region comprising LCDR1, LCDR2, LCDR3, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine implantation protein preferentially amplifies T effector cells over antibody cytokine implantation proteinsRegulatory T cells, antibodies further comprise an IgG type heavy chain and an IgG type 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-type heavy chain; comprising SEQ ID NO:37 and an IgG class light chain comprising SEQ ID NO:29, an IgG type heavy chain; comprising SEQ ID NO:39 and an IgG class light chain comprising SEQ ID NO:29, an IgG type heavy chain; comprising SEQ ID NO:37 and an IgG class light chain comprising SEQ ID NO: 38.

In one embodiment, the IL-2 molecule or fragment thereof is implanted into V _H Wherein the IL-2 molecule is a mutein. In one embodiment, the IL-2 molecule or fragment thereof is implanted into V _H Wherein the IL-2 molecule is a mutein. In one embodiment, the IL-2 molecule or fragment thereof is implanted into V _H Wherein the IL-2 molecule is a mutein. In one embodiment, the IL-2 molecule or fragment thereof is implanted into V _L Wherein the IL-2 molecule is a mutein. In one embodiment, the IL-2 molecule or fragment thereof is implanted into V _L Wherein the IL-2 molecule is a mutein. In one embodiment, the IL-2 molecule or fragment thereof is implanted into 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, at or near the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine implant protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL2 sequence does not shift the CDR sequence reading frame. In some embodiments, the antibody cytokine implant protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL-2 sequence replaces all or part of the CDR sequence. The substitution of the IL-2 molecule may be at the N-terminal region of the CDR, at the middle region of the CDR or at or near the C-terminal region of the CDR. Substitutions of IL-2 molecules may be as few as one or two amino acids of a CDR sequence or the entire CDR sequence.

In some embodiments, the IL-2 molecule is directly implanted into the CDR without a peptide linker, wherein there are no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, IL-2 molecules are indirectly implanted into the CDR through 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 certain instances, 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 its entirety.

In one embodiment, the antibody cytokine implant protein comprises a sequence selected from the group consisting of SEQ ID NOs: 16. SEQ ID NO: 19. SEQ ID NO:22 and SEQ ID NO: HCDR1 of 25. In one embodiment, the antibody cytokine implant 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 one embodiment, the antibody cytokine implant protein comprises a sequence selected from the group consisting of SEQ ID NOs: 17. SEQ ID NO: 20. SEQ ID NO:23 and SEQ ID NO: HCDR2 of 26. In one embodiment, the antibody cytokine implant 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 one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:28, V of the amino acid sequence of 28 _H A zone. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO: 29. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:36, V of the amino acid sequence of 36 _L A zone. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:37, and a light chain of the amino acid sequence of 37. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:28, V of the amino acid sequence of 28 _H A region comprising SEQ ID NO:36, V of the amino acid sequence of 36 _L A zone. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:29 and a heavy chain region comprising the amino acid sequence of SEQ ID NO:37, and a light chain region of the amino acid sequence of 37. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:29 and a heavy chain region comprising the amino acid sequence of SEQ ID NO:39, and a light chain region of an amino acid sequence of seq id no. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:38 and a heavy chain region comprising the amino acid sequence of SEQ ID NO:37, and a light chain region of an amino acid sequence of seq id no. In one embodiment, the antibody cytokine implant protein comprises: comprising SEQ ID NO:38 and a heavy chain region comprising the amino acid sequence of SEQ ID NO:39, and a light chain region of an amino acid sequence of seq id no. In one embodiment, the antibody cytokine implant protein comprises an igg.il2f71a.h1 or igg.il2r67a.h1 or variant, derivative or fragment thereof, or a protein having a conservative amino acid substitution thereof or 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 one embodiment, the antibody component of the antibody cytokine implants described herein comprises an immunoglobulin sequence, framework sequence, or CDR sequence of palivizumab. In some embodiments, the antibody cytokine implant proteins described herein have a serum half-life that is longer than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or comparable molecules. In one embodiment, the antibody cytokine implant protein described herein has a sequence as shown in table 3.

Table 3: exemplary palivizumab antibody-IL-2 implant 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 Th2T cells as well as by eosinophils, basophils, and obesity cells. IL-4 modulation initiationHelper T cells (Th 0 cells) differentiate into Th2T cells. Steinke and Borish, respir.Res.2001,2,66-70. Upon activation of IL-4, th2T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression and induces B cell type switching to IgE and IgG expression ₁ . Recombinant human IL-4 suitable for use in the present invention is available from a number of suppliers including Prospec-Tany Technoge Ltd. (Doberoren, N.J.) and ThermoFisher Scientific, inc. (Woltherm, massachusetts, U.S.A.), human IL-15 recombinant protein, product number Gibco CTP 0043. 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-4" (also referred to herein as "IL 4") refers to a cytokine called interleukin 4, which is produced by Th2T cells as well as by eosinophils, basophils, and obesity cells. IL-4 regulates the differentiation of naive helper T cells (Th 0 cells) into Th2T cells. Steinke and Borish, respir.Res.2001,2,66-70. Upon activation of IL-4, th2T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression and induces B cell type switching to IgE and IgG expression ₁ . Recombinant human IL-4 suitable for use in the present invention is available from a number of suppliers including Prospec-Tany Technoge Ltd. (Doberoren, N.J.) and ThermoFisher Scientific, inc. (Woltherm, massachusetts, U.S.A.), human IL-15 recombinant protein, product number Gibco CTP 0043. The amino acid sequences of recombinant human IL-4 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 5).

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 stimulates the development of T cells. IL-7 binds to the IL-7 receptor (heterodimer consisting of IL-7 receptor alpha and common gamma chain receptors), 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 Technoge Ltd. (Doberoren, N.J. (product No. CYT-254) and ThermoFisher Scientific, inc. (Woltherm, massachusetts, U.S.A.), human IL-15 recombinant protein, product No. Gibco PHC 0071. Recombinant human IL-7 suitable for use in the present invention is available from a number of suppliers including Prospec-Tany Technoge Ltd. (Doberoren, N.J. (product No. CYT-254) and ThermoFisher Scientific, inc. (Woltherm, massachusetts, U.S.A.), human IL-15 recombinant protein, product No. Gibco PHC 0071. The amino acid sequences of recombinant human IL-7 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 6).

The term "IL-15" (also referred to herein as "IL-15") refers to a T-cell growth factor known as interleukin-15, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biological analogs and variants thereof. IL-15 is described, for example, in Fehniger and Caligiouri, blood 2001,97,14-32, the disclosure of which is incorporated herein by reference in its entirety. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) and has a molecular weight of 12.8kDa. Recombinant human IL-15 is available from a number of suppliers including Prospec-Tany Technoge Ltd. (Doberoren Rake, new Jersey, U.S.A. (product number CYT-230-b) and ThermoFisher Scientific, inc. (Woltherm, massachusetts, U.S.A.) (human IL-15 recombinant protein, product number 34-8159-82). The amino acid sequences of recombinant human IL-15 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 7).

The term "IL-21" (also referred to herein as "IL 21") refers to pleiotropic cytokine proteins known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biological analogs and variants thereof. IL-21 is described, for example, in Spolski and Leonard, nat. Rev. Drug. Disc.2014,13,379-95, the disclosures of which are incorporated herein by reference in their entirety. IL-21 is composed primarily of natural killer T cells and activated human CD4 ⁺ T cell production. Recombinant human IL-21 is a single 132 amino acid containingNon-glycosylated polypeptide chain with a molecular weight of 15.4kDa. Recombinant human IL-21 is available from a number of suppliers including Prospec-Tany Technoge Ltd. (Doberoren, N.J. (product No. CYT-408-b) and ThermoFisher Scientific, inc. (Woltherm, massachusetts, U.S.A.) (human IL-21 recombinant protein, product No. 14-8219-80). The amino acid sequences of recombinant human IL-21 suitable for use in the present invention are given in Table 2 (SEQ ID NO: 8).

When an "anti-tumor effective amount", "tumor inhibiting effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered can be determined by a physician taking into account the age, weight, tumor size, infection or metastasis extent of the subject, and the differences in the patient's (subject's) condition. Generally, a pharmaceutical composition comprising tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) as described herein can be 10 ⁴ To 10 ¹¹ Cells/kg body weight (e.g. 10 ⁵ To 10 ⁶ 、10 ⁵ To 10 ¹⁰ 、10 ⁵ To 10 ¹¹ 、10 ⁶ To 10 ¹⁰ 、10 ⁶ To 10 ¹¹ 、10 ⁷ To 10 ¹¹ 、10 ⁷ To 10 ¹⁰ 、10 ⁸ To 10 ¹¹ 、10 ⁸ To 10 ¹⁰ 、10 ⁹ To 10 ¹¹ Or 10 ⁹ To 10 ¹⁰ Cell/kg body weight), including all whole values within these ranges. TIL (in some cases involving genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these doses. TIL (including in some cases genetically engineered TIL) can be administered using infusion techniques well known in immunotherapy (see, e.g., rosenberg et al, new eng.j. Med.1988,319, 1676). The optimal dosage and treatment regimen for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

The terms "hematological malignancy," "hematological 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). Hematological malignancies are also known as "liquid tumors". Hematological 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 hematological malignancies that affect B cells.

The term "liquid tumor" refers to an abnormal mass of cells having a fluid nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TIL obtained from liquid tumors is also referred to herein as bone Marrow Infiltrating Lymphocytes (MILs). TIL obtained from liquid tumors (including liquid tumors in the peripheral blood circulation) is also referred to herein as PBL. The terms MILs, TIL and PBL are used interchangeably herein and are only the differences in the tissue types from which the cells are derived.

The term "microenvironment" as used herein may refer to the entire solid or hematological tumor microenvironment or may refer to a separate subset of cells in the microenvironment. Tumor microenvironment as used herein refers to a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical stimuli that "promote tumor transformation, support tumor growth and invasion, protect tumors from host immunity, encourage therapeutic resistance, and provide dominant metastatic growth space" as described by Swartz et al, cancer res, 2012,72,2473. Although tumors express antigens that should be recognized by T cells, the immune system rarely undergoes tumor clearance due to the immunosuppressive effects of the microenvironment.

In one embodiment, the invention includes a method of treating cancer using 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 one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (27 and 26 days prior to TIL infusion) and fludarabine 25mg/m ² Day 5 (days 27 to 23 before TIL infusion). At the position ofIn one embodiment, following non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), the patient receives an intravenous infusion of 720,000IU/kg intravenous IL-2 every 8 hours to physiological tolerance.

Experiments have found that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by clearing regulatory T cells and competing immune system elements ("cytokine libraries"). Thus, some embodiments of the invention subject the patient to a lymphocyte depletion step (sometimes also referred to as "immunosuppressive conditioning") 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 sufficient to affect the intended application, including but not limited to, the treatment of a disease. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the subject and disease condition to be 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., reduce platelet adhesion and/or cell migration). The precise dosage will vary depending upon the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the tissue to which it is to be administered, and the physical delivery system in which the compound is to be carried.

The term "treatment" and similar terms refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic, i.e. complete or partial prophylaxis of the disease or symptoms thereof, and/or the effect may be therapeutic, i.e. partial or complete cure of the disease and/or adverse effects of the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (particularly a human) and includes: (a) Preventing the disease from occurring in a subject who is susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., stopping the development or progression of the disease; and (c) alleviating the disease, i.e., causing regression of the disease and/or alleviating one or more symptoms of the disease. "treating" also encompasses delivering an agent to provide a pharmacological effect even in the absence of a disease or a condition. For example, "treating" encompasses delivering a composition that can elicit an immune response or confer immunity in the absence of a disease condition, such as a vaccine for example.

When the term "heterologous" is used to refer to a portion of a nucleic acid or protein, it is indicated that the nucleic acid or protein comprises two or more subsequences that do not have the same relationship in nature. For example, nucleic acids are typically recombinantly produced and have two or more sequences from unrelated genes arranged to make new functional nucleic acids, e.g., a promoter from one source and a coding region from another source, or a coding region from a different source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that do not have the same relationship in nature (e.g., fusion proteins).

When referring to two or more nucleic acids or polypeptides, the terms "sequence identity", "percent identity" and "percent sequence identity" (or synonymous terms 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 the two or more sequences or subsequences are compared and aligned (if necessary, interspersed) for highest correspondence and without 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 and can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs that can determine percent sequence identity include, for example, BLAST suite programs available from the national center for Biotechnology information BLAST website of the U.S. 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, san Jose, calif.) or MegAlign (available from DNASTAR) are other publicly available alignment software programs. One skilled in the art can determine the appropriate parameters for a particular alignment software to maximize alignment. In some embodiments, the built-in 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 in that there is more than one substitution, removal, and/or addition at some position within or adjacent to the amino acid sequence of the reference antibody. More than one conservative substitution may be included in the amino acid sequence of the variant as compared to the amino acid sequence of the reference antibody. Conservative substitutions may involve, for example, substitution of similarly 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.

"tumor infiltrating lymphocytes" or "TILs" herein refer to a population of cells that are originally obtained as white blood cells but which leave the subject's blood stream and migrate into the tumor. TIL includes but is not limited to CD8 ⁺ Cytotoxic T cells (lymphocytes), th1 and Th17CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TIL includes primary TIL and secondary TIL. "Primary TILs" are those TILs obtained from patient tissue samples (sometimes referred to as "freshly collected") as outlined herein, while "secondary TILs" are any population of expanded or proliferated TIL cells described herein, including, but not limited to, subject TILs, expanded TILs ("REP TILs"), and "REP TILs" as discussed herein. The reREP TIL may include, for example, a second amplified TIL or a second additional amplified TIL (e.g., those described in step D of fig. 8, including a TIL called a reREP TIL).

TIL can generally be defined via biochemistry (using cell surface markers) or functionality (through its ability to infiltrate tumors and affect treatment). TIL can generally be categorized by the expression of 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 a solid tumor after reintroduction into a patient. TIL may be further characterized by efficacy-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 effective.

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

The term "RNA" defines a molecule comprising at least one ribonucleotide residue. The term "ribonucleotide" defines 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 such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, removal, substitution, and/or alteration of one or more nucleotides. The nucleotides of 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, coating agents, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and inert ingredients. Such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for use with the active pharmaceutical ingredient are well known in the art. Unless any known 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 (e.g., other drugs) may also be incorporated into the compositions and methods.

The term "about" or "approximately" means within a statistically significant range of numerical values. The 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 term "about" or "approximately" encompasses an allowable deviation depending on the particular research system and can be readily appreciated by one of ordinary skill in the art. Moreover, the terms "about" and "approximately" as used herein mean that the dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like and 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 "approximately," whether or not so explicitly stated. It has been noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangement.

When the transitional terms "comprising," "consisting essentially of … …," and "consisting of … …" are used in the original and modified forms of the appended claims, they define what additional requesting elements or steps (if any) are not recited in the scope of the claims. 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 … …" excludes any element, step or material not specified in the claims, in the latter case excluding impurities normally associated with the specified material. The term "consisting essentially of … …" limits the scope of the claims to those indicated as elements, steps or materials and does not materially affect the basic and novel characteristics of the claimed invention. All compositions, methods and kits embodying the invention described herein may be more specifically defined in alternative embodiments by any transitional term "comprising," consisting essentially of … …, "and" consisting of … ….

The term "antibody" refers to an intact immunoglobulin and any antigen-binding fragment ("antigen-binding portion") or single chain thereof. "antibody" further refers to a glycoprotein or antigen binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V _H ) And a heavy chain constant region. The heavy chain constant region comprises three domains (CH 1, CH2 and CH 3). Each light chain comprises a light chain variable region (abbreviated herein as V _L ) And a light chain constant region. The light chain constant region comprises a domain (C _L ). Anti-cancer agentV of body _H And V _L Regions may be further subdivided into regions of high variation known as Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs) and interspersed with regions that are more conserved known as Framework Regions (FR). Each V is _H And V _L Consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-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 epitopes. 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, the antigen is a molecule capable of being bound by an antibody or TCR if presented by a Major Histocompatibility Complex (MHC) molecule. The term "antigen" as used herein also encompasses T cell epitopes. In addition, the antigen can be recognized by the immune system. In some embodiments, the antigen is capable of inducing a humoral or cellular immune response that results in activation of B lymphocytes and/or T lymphocytes. In certain instances, this may require that the antigen contain or be linked to a Th cell epitope. Antigens may also have more than one epitope (e.g., B and T epitopes). In some embodiments, the antigen will preferably react with its corresponding antibody or TCR in general in a highly specific and selective manner and not with numerous 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 prepared using knowledge and techniques in the art, i.e., injection of the test subject with the appropriate antigen and subsequent isolation of hybridomas expressing antibodies having the desired sequence or functional characteristics. DNA encoding a monoclonal antibody can be readily isolated and sequenced using well-known procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibody). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell, such as an escherichia coli (e.coli) cell, a monkey COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell, that does not otherwise produce immunoglobulins, to obtain synthesis of monoclonal antibodies in the recombinant host cell. Recombinant production of antibodies will be described in more detail below.

The term "antigen binding site" or "antigen binding fragment" (or simply "antibody portion" or "fragment") of an antibody as used herein refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of antibodies can be performed by fragments of full length antibodies. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) Fab fragment, from V _L 、V _H 、C _L And a monovalent fragment consisting of a CH1 domain; (ii) A F (ab') 2 fragment comprising two bivalent fragments of a Fab fragment linked by a disulfide bond of the hinge region; (iii) From V _H And a Fd fragment consisting of a CH1 domain; (iv) From V of a single arm of an antibody _L And V _H Fv fragments consisting of domains; (v) Domain antibodies (dAb) fragments (Ward et al Nature,1989,341,544-546) which can be derived from V _H Or V _L Domain composition; and (vi) an isolated Complementarity Determining Region (CDR). In addition, although Fv fragment two domains V _L And V _H Encoded by separate genes, they can be joined by synthetic linkers using recombinant methods so that they can be prepared as a single protein chain, where V _L And V _H Regions pair to form monovalent molecules known as single chain Fv (scFv); see, e.g., bird et al, science 1988,242,423-426; and Huston et al, proc.Natl. Acad. Sci. USA 1988,85,5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of the antibody. These antibody fragments are obtained using known techniques known to those skilled in the art, and the effect of the fragments is screened in the same manner as for intact antibodies. In one embodiment, the scFv protein domain comprises V _H Part and V _L Part(s). ScFv (field-effect transistor)The molecule is denoted as V _L -L-V _H (if V _L The domain being the N-terminal part of an scFv molecule) or V _H -L-V _L (if V _H The domain is the N-terminal part of the scFv molecule). Methods for making scFv molecules and designing suitable peptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, r.raag and m.whitlow, "Single Chain fvs." FASEB Vol 9:73-80 (1995) R.E.bird and B.W.walker, single Chain Antibody Variable Regions, TIBTECH, vol 9:132-137 (1991), the disclosure of which is incorporated herein by reference in its entirety.

The term "human antibody" as used herein is intended to include antibodies in which the framework and CDR regions of the variable regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are 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 formed by random or site-directed mutagenesis in vitro or introduced by in vivo somatic mutation). The term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted to human framework sequences.

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

The term "recombinant human antibody" as used herein includes all human antibodies produced, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from a transgenic or transchromosomal animal (e.g., mouse) of human immunoglobulin genes or from a hybridoma produced therefrom (described further below); (b) An antibody isolated from a host cell transformed to express the human antibody, such as a transfectoma (transfectoma); (c) an antibody isolated from a recombinant combinatorial human antibody library; to be used forAnd (d) antibodies produced, expressed, produced or isolated by any other means that involves splicing the human immunoglobulin gene sequence into other DNA sequences. The recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibody may be subjected to in vitro mutagenesis (or, when transgenic animals of human Ig sequences are used, the recombinant human antibody is subjected to in vivo mutagenesis), thus V of the recombinant antibody _H And V _L The amino acid sequence of the region is, although derived from and related to human germline V _H And V _L Sequences that are not naturally occurring within the human antibody germline repertoire in vivo.

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

The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody specifically binding to an antigen".

The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody and 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) are grafted to human framework sequences. Additional framework region modifications can be made within 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. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and capacity. In certain instances, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues not found in the recipient antibody or the donor antibody. These modifications were made to further refine antibody efficacy. Typically, a humanized antibody will comprise substantially all of at least one and typically two variable domains, all or substantially all of which correspond to those of a non-human immunoglobulin and all or substantially all of which are of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See Jones et al, nature 1986,321,522-525 for further details; riechmann et al, nature 1988,332,323-329; and Presta, curr.op.struct.biol.1992,2,593-596. The antibodies described herein may also be modified to employ any Fc variant known to confer improved (e.g., reduced) effector function and/or FcR binding. Fc variants may include, for example, international patent application publication nos. WO 1988/07089 A1, WO 1996/14339 A1, WO 1998/05787 A1, WO 1998/23289 A1, WO 1999/51642 A1, WO 99/58572 A1, WO 2000/09560 A2, WO 2000/32767 A1, WO 2000/42072 A2, WO 2002/44215 A2, WO 2002/060919 A2, WO 2003/074569 A2, WO 2004/016750 A2, WO 2004/029207 A2, WO 2004/035752 A2, WO 2004/0635351 A2, WO 2004/074455 A2, WO 2004/099249 A2, WO 2005/040217 A2, WO 2005/070963 A1, WO 2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 A1, WO 2006/047350 A2 and WO 2006/085967 A2; and U.S. Pat. nos. 5,648,260;5,739,27;5,834,250;5,869,046;6,096,871;6,121,022;6,194,551;6,242,195;6,277,375;6,528,624;6,538,124;6,737,056;6,821,505;6,998,253 and 7,083,784; the disclosure of which is incorporated herein by reference in its entirety.

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 "diabody" is a small antibody fragment having two antigen binding sites. The fragment comprises a light chain variable domain (V _L ) Linked heavy chain variable domains (V _H )(V _H -V _L Or V _L -V _H ). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with the complementary domain of the other strand and create two antigen binding sites. Diabodies are described, for example, in European patent No. EP 404,097, international patent publication No. WO 93/11161; and 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 result in the removal of more than one variable region framework glycosylation site to thereby remove glycosylation at that site. Non-glycosylation 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, such as low fucosylation antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structure, can be prepared. Such altered glycosylation patterns have been shown to increase the ability of antibodies. Such carbohydrate modification may be accomplished, for example, by expressing the antibody in a host cell that alters the glycosylation machinery. Cells that alter the glycosylation machinery have been described in the art and can be used as host cells for expression of 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. Ms704, ms705 and Ms709FUT 8-/-cell lines were generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see, e.g., U.S. patent publication No. 2004/010704 or Yamane-Ohnuki et al, biotechnol. Bioeng.,2004,87,614-622). As another example, european patent No. 1,176,195 describes a cell line with functional disruption of FUT8 gene encoding a fucosyltransferase such that antibodies expressed in the cell line exhibit low fucosylation by reducing or eliminating α1,6 linkage-associated enzymes, and also describes a cell line with low or no enzymatic activity for adding fucose to N-acetylglucosamine bound to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). International patent publication No. WO 03/035835 describes a variant CHO cell line Lec 13 cell with reduced ability to link fucose to Asn (297) for linking carbohydrates, also resulting in low fucosylation of antibodies expressed in the host cell (see also Shields et al, J.biol. Chem.2002,277, 26733-26740). International patent publication No. WO 99/54342 describes cell lines engineered to express glycoprotein-modified glycosyltransferases, such as beta (1, 4) -N-acetylglucosaminyl transferase III (GnTIII), such that antibodies expressed in the engineered cell lines exhibit increased bisected GlcNac structure, resulting in increased antibody ADCC activity (see also Umana et al, nat. Biotech.1999,17, 176-180). Alternatively, the fucose residues of the antibodies may be cleaved using a fucosidase. For example, the fucosidase α -L-fucosidase removes fucosyl residues from the antibody as described by Tarentino et al, biochem.1975,14, 5516-5523.

"PEGylation" refers to a modified antibody (or fragment thereof) that typically reacts with a PEG, such as a reactive ester or aldehyde derivative of PEG, under conditions that cause more than one polyethylene glycol (PEG) group to become attached to the antibody or antibody fragment. PEGylation may, for example, 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). The term "polyethylene glycol" as used herein is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C) ₁ -C ₁₀ ) Alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. Quilt is covered withThe pegylated antibody may be an aglycosylated antibody. Methods of pegylation are known in the art and can be applied to the antibodies of the present invention, as described, for example, in European patent numbers EP 0154316 and EP 0401384 and U.S. Pat. No. 5,824,778, the respective disclosures of which are incorporated herein by reference in their entirety.

The term "biosimilar" refers to a biological product (including monoclonal antibodies or proteins) that is highly similar to a reference biological product licensed in the united states, and that, despite minor differences in clinically inactive components, has no clinically significant differences in product safety, purity and efficacy from the reference product. In addition, a similar biological or "bio-similar" drug is a biopharmaceutical similar to another biopharmaceutical that has been authorized for use by the European drug administration. The term "biological analogue" is also used synonymously by other national and regional authorities. A biological product or biopharmaceutical is a drug prepared from or derived from a biological source (e.g., bacteria or yeast). They 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 approved by the regulatory agency for reference to aldesleukin is "biosimilar" with or "biosimilar" with reference to aldesleukin. In europe, a similar biological or "bio-like" drug is a biopharmaceutical similar to another biopharmaceutical that has been authorized for use by the european drug administration (EMA). The relevant legal basis for similar biological applications in Europe is revised Regulation (EC) No 726/2004, clause 6 and Directive2001/83/EC, clause 10 (4), so that biological analogs in Europe may be authorized, approved or subject matter of application authorization in accordance with Regulation (EC) No 726/2004, clause 6 and Directive2001/83/EC, clause 10 (4). The original biopharmaceutical product that has been authorized may be referred to as a "reference drug" in europe. Some of the regulations considered for bio-analog products are summarized in the biopharmaceutical-like CHMP guidelines (CHMP Guideline on Similar Biological Medicinal Products). In addition, product specific criteria (including criteria related to monoclonal antibody biological analogs) are provided by EMA based on individual products and published on the website. The biosimilar as described herein may be similar to the reference drug in terms of quality characteristics, bioactivity, mechanism of action, safety profile, and/or efficacy. Furthermore, biological analogs can be used or intended for the treatment of the same instances of a reference drug. Thus, a biological analog as described herein may be considered to have quality characteristics similar or highly similar to a reference drug. Alternatively or in addition, a biological analog as described herein may be considered to have similar or highly similar biological activity to a reference drug. Alternatively or in addition, a biological analog as described herein may be considered to have similar or highly similar safety characteristics as the reference drug. Alternatively or in addition, a biological analog as described herein may be considered to have a similar or highly similar therapeutic effect as the reference drug. As described herein, the biological analogs of europe are compared to reference drugs that have been authorized for EMA. However, in some instances, biological analogs may be compared to biopharmaceuticals (non-EEA authorized "comparator") that in some studies have been obtained outside of the european economy. Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biological analog" also pertains to a biopharmaceutical that has 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 biological analogue of a protein may have an amino acid sequence that is slightly modified (including, for example, amino acid substitutions, additions and/or deletions) in the amino acid structure without significantly affecting the function of the polypeptide. A biological analog may comprise an amino acid sequence that has 97% or greater than 97% (e.g., 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of its reference drug. The biological analog 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 the difference does not result in a change in drug safety and/or efficacy. The biological analog may have the same or a different glycosylation pattern than the reference drug. Particularly (but not exclusively) if the differential treatment or intention addresses safety concerns associated with the reference drug, the biological analogs may have different glycosylation patterns. Furthermore, the biological analog may deviate from the reference drug in terms of, for example, its strength, pharmaceutical form, formulation, excipient, and/or appearance, so long as the safety and efficacy of the drug are not compromised. Biological analogs may include differences in Pharmacokinetic (PK) and/or Pharmacodynamic (PD) properties, for example, as compared to a reference drug, but are still considered sufficiently similar to a reference drug to be authorized or considered suitable for authorization. In certain instances, the biological analogs exhibit a binding profile that is different from the reference drug, wherein the different binding profile is not considered by the governing body (e.g., EMA) as a barrier authorized to resemble a biological product. The term "biological analogue" is also used synonymously by other national and regional authorities.

III, 2 nd generation TIL manufacturing Process

An exemplary family of TIL processes, called generation 2 (also called process 2A), containing some of these features is depicted in fig. 1 and 2. The embodiment of generation 2 is shown in fig. 2.

As discussed herein, the present invention may include steps relating to re-stimulating cryopreserved TILs to increase their metabolic activity and thus relative health prior to implantation into a patient, as well as methods of testing such metabolic health. As generally summarized herein, TILs are typically taken from patient samples and are manipulated to amplify their amounts prior to transplantation into a patient. In some embodiments, the TIL may optionally be genetically manipulated as discussed below.

In some embodiments, the TIL may be cryopreserved. Once thawed, they may also be re-stimulated to increase their metabolism prior to infusion into a patient.

In some embodiments, as discussed in detail below and in the examples and figures, the first amplification (including the process prior to REP and the process shown in step a of fig. 1) is shortened to 3 to 14 days and the second amplification (including the process known as REP and the process shown in step B of fig. 1) is shortened to 7 to 14 days. 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 as described in step B and step D of fig. 1) is shortened to 22 days, as discussed in detail below and in the examples and figures.

The following "step" code A, B, C, etc. refer to fig. 1 and to certain embodiments described herein. The sequence of steps below and in fig. 1 is exemplary and any combination or sequence of steps as well as additional steps, repeated steps and/or omission of steps are contemplated in the present application and methods disclosed herein.

A. Step A: obtaining a tumor sample of a patient

Generally, TIL is initially obtained from a patient tumor sample and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, 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 biopsy, core biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multi-focal sampling is used. In some embodiments, surgical resection, needle aspiration biopsy, core biopsy, small biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells include multifocal sampling (i.e., obtaining a sample from one or more tumor sites and/or locations of a patient and one or more tumors at the same location or close range). 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 hematological malignancy. The solid tumor may be lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung cancer (NSCLC).

Once obtained, the tumor sample is typically fragmented into pieces ranging from 1 to about 8mm using a sharp instrument ³ Small pieces between, and about 2 to 3mm ³ Is especially useful. In some casesIn embodiments, TIL is cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., losv-pak souvenir 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 obtained by placing the tumor in an enzyme medium and mechanically dissociating the tumor for about 1 minute, followed by 5% CO at 37 ℃ ₂ After 30 minutes of incubation, the mechanical dissociation and incubation cycle was repeated under the conditions described above until only small tissue pieces were present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using FICOLL branched hydrophilic polysaccharides to remove these cells. Alternative methods known in the art, such as those described in U.S. patent application publication 2012/0244233 A1, the disclosure of which is incorporated herein by reference in its entirety, may be used. 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 more than one dissociating (digesting) enzyme, such as, but not limited to collagenase (including collagenase of any blend or type), accutase ^TM 、Accumax ^TM Hyaluronidase, neutral protease (dispase), chymosin, chymopapain, trypsin, casein, elastase, papain, type XIV 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 from a freeze-dried enzyme. In some embodiments, the lyophilized enzyme is reconstituted with an amount of a sterile buffer (e.g., HBSS).

In certain instances, collagenase (e.g., animal-free collagenase type 1) is reconstituted with 10mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme may be 2892PZ U/vial. In some embodiments, collagenase is reconstituted with 5mL to 15mL buffer. In some embodiments, the range of the collagenase stock solution after reconstitution is from about 100PZ U/mL to about 400PZ U/mL, 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 neutral protease is reconstituted with 1mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme may be 175DMC U/vial. In some embodiments, the range of neutral pro-protein liquid after reconstitution is from about 100DMC/mL to about 400DMC/mL, such as 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, from about 110DMC/mL, from about 120DMC/mL, from about 130DMC/mL, from about 140DMC/mL, from about 150DMC/mL, from about 160DMC/mL, from about 170DMC/mL, from about 175DMC/mL, from about 180DMC/mL, from about 190DMC/mL, from about 200DMC/mL, from about 250DMC/mL, from about 300DMC/mL, from about 350DMC/mL, or from about 400DMC/mL.

In some embodiments, dnase I is reconstituted with 1mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme was 4 KU/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 zymogen fluid is variable and the concentration may need to be determined. In some embodiments, the concentration of the freeze-dried stock solution can be verified. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the measured stock solution concentration.

In some embodiments, the enzyme mixture comprises about 10.2-ul of neutral protease (0.36 DMC U/mL), 21.3. Mu.L of collagenase (1.2 PZ/mL), and 250-ul of DNase I (200U/mL) in about 4.7mL of sterile HBSS.

As indicated above, the first and second light sources,in some embodiments, the TIL is derived from a solid tumor. In some embodiments, the solid tumor is not fractured. In some embodiments, the solid tumor is not fragmented and the enzyme digestion is performed with a 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 treated with 5% CO at 37 ℃ in an enzyme mixture comprising collagenase, dnase, and hyaluronidase ₂ Digestion was performed for 1 to 2 hours. In some embodiments, the tumor is treated with 5% CO at 37 ℃ in an enzyme mixture comprising collagenase, dnase, and hyaluronidase ₂ Digestion under rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is at 37 ℃, 5% CO ₂ Digestion was carried out overnight under constant rotation. In some embodiments, the whole 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 collagenase working stock is 100mg/mL 10X working stock.

In some embodiments, the enzyme mixture comprises dnase. In some embodiments, the dnase working stock is 10,000iu/mL 10X working stock.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10-mg/mL 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, shredding includes physical shredding, including, for example, segmentation and digestion. In some embodiments, the breaking is physical breaking. In some embodiments, the fracture is a 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 one embodiment, 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 fragmented after obtaining a tumor sample, e.g., in step a (as provided in fig. 1). In some embodiments, the disruption occurs prior to cryopreservation. In some embodiments, the disruption occurs after cryopreservation. In some embodiments, the disruption occurs after the tumor is obtained and no cryopreservation is present. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed into each container for first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed into each container for first amplification. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed into each container for first amplification. In some embodiments, the plurality of fragments comprises from about 4 to about 50 fragments, each fragment having about 27mm ³ Is a volume of (c). In some embodiments, the plurality of segments comprises about 30 to about 60 segments, with a total volume of about 1300mm ³ Up to about 1500mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, the total volume of which is 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 Between them. In some embodiments, the tumor fragments are between about 1mm ³ And 8mm ³ Between them. 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 by 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 x 4mm.

In some embodiments, the tumor is resected to minimize the amount of bleeding, necrosis, and/or adipose tissue on each patch. In some embodiments, the tumor is resected to minimize the amount of bleeding tissue on each patch. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each patch. In some embodiments, the tumor is resected to minimize the amount of adipose tissue on each slice.

In some embodiments, tumor morcellation is performed to maintain tumor internal structure. In some embodiments, the performing of tumor morcellation does not include performing a sawing action using a scalpel. In some embodiments, the TIL is obtained from tumor digests. In some embodiments, tumor digests are produced by incubation in an enzyme medium such as, but not limited to, RPMI1640, 2mM Glutamax, 10mg/mL gentamicin, 30U/mL DNase, and 1.0mg/mL collagenase, followed by mechanical dissociation (GentleMACS, miltenyi Biotec, ornith, calif.). After placing the tumor in the enzyme medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be brought to a temperature of 37℃at 5%CO ₂ For 30 minutes, followed by mechanical disruption again for about 1 minute. At 37℃at 5% CO ₂ After an additional 30 minutes of incubation, the tumor may be mechanically destroyed a third time for about 1 minute. In some embodiments, if a large piece of tissue is still present after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, whether or not at 5% CO at 37 ℃ anymore ₂ For 30 minutes. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells 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.

1. Pleural effusion T cells and TIL

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cell TILs for expansion according to the processes described herein is a pleural fluid sample. In some embodiments, the sample is a pleural effusion derived sample. 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 No. US 2014/0295426, 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, such as 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 TIL-containing slurries, including, for example, ascites in the abdomen or pancreatic cyst fluid. Ascites and pleural fluids involve very similar chemical systems; both the abdomen and the lungs have mesothelial cell lines and in malignant disease in the same situation form fluids in the pleural and abdominal spaces, such fluids containing TIL in some embodiments. In some embodiments, the present disclosure exemplifies pleural fluid, and the same method may be performed using ascites or other cyst fluid containing TIL to achieve similar results.

In some embodiments, the pleural fluid is in an untreated, directly as removed from the patient. 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 step In a test tube (Veridex). In some embodiments, samples are placed into CellSave tubes immediately after collection from the patient to avoid a decrease in the number of surviving TILs. If left in untreated pleural fluid even at 4 ℃, the number of surviving TILs can be reduced to a significant extent within 24 hours. In some embodiments, the sample is placed into an appropriate collection tube within 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 into an appropriate collection tube within 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 one embodiment, the dilution is 1:10 pleural fluid to diluent. In another embodiment, the dilution is 1:9 pleural fluid vs. diluent. In another embodiment, the dilution is 1:8 pleural fluid vs. diluent. In another embodiment, the dilution is 1:5 pleural fluid to diluent. In another embodiment, the dilution is 1:2 pleural fluid to diluent. In another embodiment, the dilution is 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 into CellSave tubes immediately after collection and dilution from the patient to avoid a decrease in viable TIL that can occur to a significant extent within 24 to 48 hours if left in untreated pleural fluid even at 4 ℃. In some embodiments, the pleural fluid sample is placed into the appropriate collection tube within 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 into a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal and dilution from the patient at 4 ℃.

In yet another embodiment, the pleural fluid sample is concentrated by known means prior to the further processing step. In some embodiments, this pretreatment of pleural fluid is preferred in instances where the pleural fluid must be cryopreserved for shipment to the laboratory where the method is performed or for later 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 withdrawn from the subject and resuspending the centrifugal isolate or pellet in a buffer. In some embodiments, pleural fluid samples are 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 to allow pleural fluid to pass through the membrane but retain tumor cells. In some embodiments, the holes in the membrane may be at least 4 μm in diameter. In another embodiment, the pore diameter may be 5 μm or more than 5 μm, in any of the other embodiments 6, 7, 8, 9, or 10 μm. After filtration, cells retained by the membrane (including TIL) may be rinsed off the membrane into a suitable physiologically acceptable buffer. The 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 a mass of resuspended cells is contacted with a lysing agent that differentially lyses the non-nucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in situations where the pleural fluid contains a large number of RBCs. Suitable lysing reagents include single lysing reagents or lysing reagents and quenching reagents or lysing reagents, quenching reagents and immobilization reagents. Suitable cleavage systems are commercially available, including BD PharmLyse ^TM System (Becton Dickenson). Other cleavage systems include Versalysie ^TM System, FACSly ^TM System (Becton Dickenson), immunoprep ^TM The system or the Erythrolyse II system (Beckman Coulter, inc.) or the ammonium chloride system. In some embodiments, the lysing agent may vary with the primary requirements (phenotypic properties of TIL and TIL in the effective lysing red blood cells and retaining pleural fluid). In addition to employing a single lysing reagent, the lysing systems useful in the methods described herein may include a second reagent, such as a reagent that quenches or blocks the effect of the lysing reagent during the remaining steps of the method, such as stabilysine ^TM Reagents (Beckman Coulter, inc.). Depending on the choice of cleavage reagent or the preferred execution of the method, well known immobilization reagents may also be used.

In some embodiments, an untreated, diluted, or multiple centrifuged or treated pleural fluid sample as described herein above is cryopreserved at a temperature of about-140 ℃ prior to further treatment and/or amplification provided herein.

B. And (B) step (B): first amplification

In some embodiments, the present methods provide for obtaining a young TIL that increases replication cycle after administration to a subject/patient, thus potentially providing additional therapeutic benefits over older TILs (i.e., TILs that have been replicated more times further prior to administration to a subject/patient). Features of young TILs have been described in the literature, for example Donia et al, scand.j. Immunol.2012,75,157-167; dudley et al, clin.cancer Res.2010,16,6122-6131; huang et al, J.Immunother.2005,28,258-267; besser et al, clin.cancer Res.2013,19, OF1-OF9; besser et al, J.Immunother.2009,32,415-423; robbins et al, J.Immunol.2004,173,7125-7130; shen et al, j.immunother, 2007,30,123-129; zhou et al, J.Immunother.2005,28,53-62; and Tran et al, j.immunother, 2008,31,742-751, each of which is incorporated herein by reference in its entirety.

The diverse 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 methods of producing TILs that exhibit and increase T cell reservoir diversity. In some embodiments, the TIL obtained by the present methods exhibits increased T cell reservoir diversity. In some embodiments, the TIL obtained by the present methods exhibits increased T cell reservoir diversity compared to fresh collection of TIL and/or TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1. In some embodiments, the TIL obtained by the present methods exhibits increased T cell reservoir diversity compared to freshly collected TIL and/or TIL prepared using a method referred to as process 1C (as exemplified in fig. 5 and/or fig. 6). In some embodiments, the TIL obtained at 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 in the immunoglobulin, in the heavy chain of the immunoglobulin. In some embodiments, the diversity is in the immunoglobulin light chain in the immunoglobulin. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is 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, the expression of TCRab (i.e., tcra/β) is increased.

After splitting or digesting tumor fragments, such as described in step a of fig. 1, for example, the resulting cells are cultured in serum containing IL-2 under conditions that favor TIL but disfavor tumor and other cell growth. In some embodiments, tumor digests are incubated in medium comprising deactivated human AB serum and 6000IU/mL IL-2 in 2mL wells. This primary cell population is cultured for a period of days (typically 3 to 14 days), resulting in a typical cell population of about 1X 10 ⁸ A host population of individual host TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, resulting in a typical cell population of about 1 x 10 ⁸ A host population of individual host TIL cells. In some embodiments, this primary cell population is cultured for a period of 10 to 14 days, resulting in a typical cell population of about 1×10 ⁸ A host population of individual host TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a typical cell population of about 1×10 ⁸ A host TIL population of host TIL cells.

In a preferred embodiment, amplification of TIL may be performed using an initial subject TIL amplification step (e.g., such as those described in step B of fig. 1, which may include a process prior to termed REP) as described below, followed by a second amplification (step D, including a process termed rapid amplification protocol (REP) step) as described below, followed by an optional cryopreservation, and followed by a second step D (including a process termed restimulation REP step) as described below and herein. TIL obtained from this process can optionally be identified as described herein with phenotypic characteristics and metabolic parameters.

In embodiments where the TIL culture starts in a 24-well plate (e.g., using Costar 24-well flat bottom cell culture plate (Corning Incorporated, corning, N.Y.), each well may be seeded with 1X 10 ⁶ Each tumor digested cell or one tumor fragment was placed in 2mL Complete Medium (CM) containing IL-2 (6000 IU/mL; chiron Corp., emeryverer, calif.). In some embodiments, the tumor fragments are between about 1mm ³ And 10mm ³ Between them.

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. At the start of the culture, the culture was started with a volume of 40mL and 10cm ² In embodiments of gas permeable flasks (e.g., G-Rex10; wilson Wolf Manufacturing, new Britton, minnesota) with a gas permeable silicon bottom (FIG. 1), each flask is loaded with 10 to 40X 10 ⁶ Individual surviving tumor digests cells or 5 to 30 tumor fragments were in 10 to 40mL of CM containing IL-2. Both G-Rex10 and 24 well plates were incubated at 37℃with 5% CO ₂ Half of the medium was removed and replaced with fresh CM and IL-2 5 days after initiation of culture, and half of the medium was replaced every 2 to 3 days after day 5.

After preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor TIL but disfavor tumor and other cell growth. In some embodiments, tumor digests are incubated in medium comprising deactivated human AB serum (or in the presence of a population of aAPC cells in some cases as outlined herein) and 6000IU/mL IL-2 in 2mL wells. This primary cell population is cultured for a period of days (typically 10 to 14 days), resulting in a typical cell population of about 1 x 10 ⁸ A host TIL population of host TIL cells. In some embodiments, the growth medium during the first amplification comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1mg vial of IL-2 stock solution has 20 to 30X 10 ⁶ IU/mg specific activity. In some embodiments, the 1mg vial of IL-2 stock solution has a 20X 10 ⁶ IU/mg specific activity. In some embodiments, the 1mg vial of IL-2 stock solution has a 25X 10 ⁶ IU/mg specific activity. In some embodiments, the 1mg vial of IL-2 stock solution has a 30X 10 ⁶ IU/mg specific activity. In some embodiments, the IL-2 stock solution has a length of 4 to 8X10 ⁶ The final concentration of IU/mg IL-2. In some embodiments, the IL-2 stock solution has a length of 5 to 7X 10 ⁶ The final concentration of IU/mg IL-2. In some embodiments, the IL-2 stock solution has a concentration of 6X10 ⁶ The final concentration of IU/mg IL-2. In some embodiments, IL-2 stock solution is prepared as described in example 5. In some embodiments, the first amplification medium comprises about 10,000IU/mL of IL-2, about 9,000IU/mL of IL-2, about 8,000IU/mL of IL-2, about 7,000IU/mL of IL-2, about 6000IU/mL of IL-2, or about 5,000IU/mL of IL-2. In some embodiments, the first amplification medium comprises from about 9,000IU/mL IL-2 to about 5,000IU/mL IL-2. In some embodiments, the first amplification medium comprises from about 8,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the first amplification medium comprises from 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 one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, 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 of IL-2. In one embodiment, 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 from about 500IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises from about 400IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises from 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 one embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the first amplification medium comprises about 20IU/mL of IL-21, about 15IU/mL of IL-21, about 12IU/mL of IL-21, about 10IU/mL of IL-21, about 5IU/mL of IL-21, about 4IU/mL of IL-21, about 3IU/mL of IL-21, about 2IU/mL of IL-21, about 1IU/mL of IL-21, or about 0.5IU/mL of IL-21. In some embodiments, the first amplification medium comprises from about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises from about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises from about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises from about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises from 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 one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1IU/mL IL-21.

In one embodiment, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL OKT-3 antibody. In one embodiment, 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 of the OKT-3 antibody. In one embodiment, 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, 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, and 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 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 one or more TNFRSF agonists, one or more TNFRSF agonists comprising 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. At the start of the culture, the culture was started with a volume of 40mL and 10cm ² In embodiments of gas permeable flasks (e.g., G-Rex10; wilson Wolf Manufacturing, new Britton, minnesota) with a gas permeable silicon bottom (FIG. 1), each flask is loaded with 10 to 40X 10 ⁶ Individual surviving tumor digests cells or 5 to 30 tumor fragments were in 10 to 40mL of CM containing IL-2. Both G-Rex10 and 24 well plates were incubated at 37℃with 5% CO ₂ 5 days after initiation of culture, half of the culture medium was removed and replaced with fresh CM and IL-2,half of the medium was changed every 2 to 3 days after day 5. In some embodiments, the 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, for example, those described in step B of fig. 1, which may include those sometimes referred to as before REP) process is shortened to 3 to 14 days, as discussed in the examples and figures. In some embodiments, the first amplification (including, for example, those described in step B of fig. 1, which may include those sometimes referred to as before REP) is shortened to 7 to 14 days, as discussed 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, as discussed in, for example, 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 employed 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 process according to step B of FIG. 1 and as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed 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 process according to step B of FIG. 1 and as described herein.

In some embodiments, the first amplification (including the process prior to what is known as REP; e.g., according to step B of FIG. 1) is shortened to 3 to 14 days, as discussed in the examples and figures. 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 (e.g., according to step B 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.

1. Cytokines and other additives

The amplification methods described herein generally use media with high doses of cytokines (specifically IL-2), as known in the art.

Alternatively, it is additionally possible to use a combination of cytokines for rapid amplification and or second amplification of TIL, as described in U.S. patent application publication No. US 2017/0107490 A1, the disclosure of which is incorporated herein by reference in its entirety, for two or more combinations of IL-2, IL-15 and IL-21. 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, wherein the latter has particular utility in many embodiments. The use of a combination of cytokines is particularly advantageous for lymphocyte production, particularly T cells as described therein.

In one embodiment, step B may also comprise adding OKT-3 antibody or moromiab to the medium as described elsewhere herein. In one embodiment, step B may also include adding a 4-1BB agonist to the medium as described elsewhere herein. In one embodiment, step B may also comprise adding an OX-40 agonist to the medium as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator activated receptor gamma coactivator I-alpha-agonists, including proliferation 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. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference in its entirety.

C. Step C: transition from first to second amplification

In certain instances, a population of subject TILs obtained from the first amplification, including, for example, a population of TILs obtained from step B, e.g., as shown in fig. 1, can be immediately cryopreserved using the procedure discussed herein below. Alternatively, the TIL population obtained from the first amplification (referred to as the second TIL population) may be subjected to a second amplification (which may include an amplification sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, in a situation in which a genetically modified TIL is to be used in therapy, a first population of TILs (sometimes referred to as a subject population of TILs) or a second population of TILs (which in some embodiments may include a population referred to as a REP population of TILs) 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., step B shown in fig. 1) is stored until phenotypes are determined for selection. In some embodiments, TIL obtained from a first amplification (e.g., step B shown in fig. 1) is directly subjected to a second amplification without storage. 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 the first amplification to the second amplification occurs 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 the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 3 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 4 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 4 days to 10 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 7 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 14 days after the disruption occurs.

In some embodiments, the transition from the first amplification to the second amplification occurs 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 the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 1 day to 14 days after the disruption occurs. 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 occurs from 3 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 4 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 5 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 6 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 7 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 8 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 9 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 10 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 11 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 12 days to 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 13 days and 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 14 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 1 day to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 2 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 3 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 4 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 5 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 6 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 7 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 8 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 9 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 10 days to 11 days after the disruption occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 11 days after the disruption occurs.

In some embodiments, the TIL is not stored after the first amplification and before the second amplification, and the TIL is directly subjected to the second amplification (e.g., in some embodiments, is not stored during the transition from step B to step D as shown in fig. 1). In some embodiments, the transition occurs 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 TIL cell population is expanded in number after collection and initial subject treatment (e.g., step a and step B shown in fig. 1) and transformation (referred to as step C). 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 shown in step D of fig. 1. The second amplification is typically accomplished in a gas-permeable vessel using a medium that contains some components, including feeder cells, a cytokine source, and anti-CD 3 antibodies.

In some embodiments, the second amplification of TIL or second TIL amplification (which may include amplification sometimes referred to as REP; and the process shown in step D of FIG. 1) may be performed using any TIL culture flask or vessel known to those skilled 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 one embodiment, 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 shown as step D of FIG. 1). For example, TIL may 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 stimulation may include, for example, anti-CD 3 antibodies such as OKT3 at about 30ng/mL, mouse monoclonal anti-CD 3 antibodies (available from Ortho-McNeil (latin, new jersey) or Miltenyi Biotech (obu, california)), or UHCT-1 (available from BioLegend, san diego, california). TIL can be amplified by including more than one antigen of cancer (including antigenic portions thereof, such as epitopes) during the second amplification to induce further in vitro stimulation of TIL, which can optionally be expressed from a vector, e.g., human white blood cell 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 such as 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 HLA-A2 expressing antigen presenting cells. Alternatively, the TIL may be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of the second amplification. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, 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 of IL-2. In one embodiment, 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 between 8000IU/mL of IL-2.

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 procedure according to step D of FIG. 1 and as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is used 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 step D of 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 occurs in supplemented cell culture medium. In some embodiments, the supplemented 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 occurs 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 from 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 from 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 one embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the second amplification medium comprises about 20IU/mL of IL-21, about 15IU/mL of IL-21, about 12IU/mL of IL-21, about 10IU/mL of IL-21, about 5IU/mL of IL-21, about 4IU/mL of IL-21, about 3IU/mL of IL-21, about 2IU/mL of IL-21, about 1IU/mL of IL-21, or about 0.5IU/mL of IL-21. In some embodiments, the second amplification medium comprises from about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises from about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises from about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises from about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises from 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 one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TIL to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In one embodiment, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1 and 50 and 1 to 300. In one embodiment, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1 to 100 and 1 to 200.

In one embodiment, REP and/or second expansion is performed in culture flasks, and bulk TIL is mixed with 100 or 200 fold excess of deactivated feeder cells, 30mg/mL OKT3 anti-CD 3 antibody and 3000IU/mL IL-2 in 150mL medium. Replacement medium (2/3 medium is replaced, typically by aspiration of fresh medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable vessels 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 discussed in the examples and figures. In some embodiments, the second amplification is shortened to 11 days.

In one embodiment, REP and/or second amplification can be performed using a T-175 flask and a gas-permeable bag as previously described (Tran et al, J.Immunothe.2008, 31,742-51; dudley et al, J.Immunothe.2003, 26,332-42) or a gas-permeable culture vessel (G-Rex flask). In some embodiments, the second amplification (including amplification known as rapid amplification) is performed in a T-175 flask, which may be suspended in 150mL of medium at about 1X 10 ⁶ TIL was added to each T-175 flask. TIL can be cultured in a 1 to 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 And (3) incubating. Half of the medium can be exchanged on day 5 with 50/50 medium containing 3000IU/mL IL-2. In some embodiments, cells from two T-175 flasks may be combined in a 3L bag on day 7 and 300mL AIM V containing 5% human AB serum and 3000IU/mL IL-2 added to 300mL TIL suspension. Cell number in each bag daily or every two daysCalculated once, fresh medium was added to maintain cell counts between 0.5 and 2.0X10 ⁶ Individual cells/mL.

In one embodiment, the second amplification (which may include amplification called REP, and those referred to in step D of FIG. 1) may be performed in 500mL capacity gas-permeable flasks (G-Rex 100, available from Wilson Wolf Manufacturing Corporation, new Britton, minnesota, USA) with a 100cm gas-permeable silicon bottom, 5X 10 ⁶ Or 10X 10 ⁶ TIL can be cultured with PBMC 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℃with 5% CO ₂ And (3) incubating. On day 5, 250mL of supernatant may be removed and placed in a centrifuge bottle and centrifuged at 1500rpm (491 Xg) for 10 minutes. The TIL pellet can be resuspended in 150mL of fresh medium containing 5% human AB serum, 3000IU/mL IL-2, and added back to the original G-Rex 100 flask. When TIL is continuously amplified in G-Rex 100 flasks, TIL in each G-Rex 100 can be suspended in 300mL of medium present in each flask on day 7, and the cell suspension can be split into 3 100mL aliquots that can be used to inoculate 3G-Rex 100 flasks. 150mL of AIM-V containing 5% human AB serum and 3000IU/mL of IL-2 can then be added to each flask. G-Rex 100 flasks can be incubated at 37℃with 5% CO ₂ After 4 days 150mL of AIM-V containing 3000IU/mL of IL-2 can be added to each G-REX 100 flask. Cells may be collected on day 14 of culture.

In one embodiment, the second amplification (including amplification called REP) is performed in culture flasks, wherein the subject TIL is mixed with 100 or 200-fold excess of deactivated feeder cells, 30mg/mL OKT3 anti-CD 3 antibody, and 3000IU/mL IL-2 in 150mL medium. In some embodiments, the replacement medium is performed until the cells are transferred to an alternative growth chamber. In some embodiments, 2/3 of the medium is replaced by pumping fresh medium. In some embodiments, the alternative growth chamber includes a G-REX flask and a gas permeable container as discussed more fully below.

In one embodiment, a second amplification (including an amplification called REP) is performed and further comprises 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 2016/0010058 A1, the disclosure of which is incorporated herein by reference in its entirety, may be used to select TILs that are excellent in tumor reactivity.

Alternatively, the cell viability assay may be performed after the second amplification (including amplification known as REP amplification) using standard assays known in the art. For example, trypan blue exclusion assays can be performed on samples of bulk TIL that selectively mark dead cells and allow viability assessment. In some embodiments, TIL samples can be counted and assayed for viability using a Cellometer K2 automated cell counter (Nexcelom Bioscience, larens, ma). In some embodiments, viability is determined according to a standard cell counter K2Image Cytometer automated cell counter protocol.

In some embodiments, the second amplification of TIL (including amplification referred to as REP) may be performed using T-175 flasks and gas-permeable bags (Tran et al 2008,J Immunother, 31:742-751 and Dudley et al, 2003,J Immunother, 26, 332-342) or gas-permeable G-Rex flasks 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 a T-175 flask, about 1X 10 ⁶ Each TIL was suspended in 150mL of medium and added to each T-175 flask. TIL was cultured with irradiated (50 Gy) allogeneic PBMC as "feeder" cells at a ratio of 1 to 100, and cells were cultured in a 1 to 1 mixture (50/50 medium) supplemented with 3000IU/mL IL-2 and 30ng/mL anti-CD 3 CM to AIM-V medium. T-175 flask was incubated at 37℃with 5% CO ₂ And (3) incubating. In some embodiments, half of the medium is replaced on day 5 with 50/50 medium containing 3000IU/mL IL-2. In some embodiments, cells from 2T-175 flasks were combined in a 3L bag on day 7 and 300mL AIM-V containing 5% human AB serum and 3000IU/mL IL-2 was added to 300mL TIL suspension. In each bagThe number of cells may be counted daily or every second day, fresh medium may be added to maintain the cell count between about 0.5 and about 2.0X10 ⁶ Between individual cells/mL.

In some embodiments, the second amplification (including what is known as REP) has a 100cm capacity at 500mL ² The culture was carried out in a flask (G-Rex 100, wilson Wolf) with a permeable silicon bottom (FIG. 1), about 5X 10 ⁶ Or 10X 10 ⁶ The TILs were incubated with irradiated allogeneic PBMC at a ratio of 1 to 100 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 5% CO at 37 ℃ ₂ And (3) incubating. In some embodiments, 250mL of supernatant is removed on day 5 and placed into a centrifuge bottle and centrifuged at 1500rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150mL of fresh 50/50 medium containing 3000IU/mL IL-2 and added back to the original G-Rex 100 flask. In an embodiment in which TILs are serially amplified in G-Rex 100 flasks, TILs in each G-Rex 100 were suspended in 300mL of medium present in each flask on day 7, and the cell suspension was split into three 100mL aliquots for inoculation of 3G-Rex 100 flasks. 150mL of AIM-V containing 5% human AB serum and 3000IU/mL of IL-2 was then added to each flask. G-Rex 100 flasks were incubated at 5% CO at 37 ℃ ₂ After 4 days 150mL of AIM-V containing 3000IU/mL of IL-2 was added to each G-Rex 100 flask. Cells were collected on day 14 of culture.

The diverse 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 methods of producing TILs that exhibit and increase T cell reservoir diversity. In some embodiments, the TIL obtained by the present methods exhibits increased T cell reservoir diversity. In some embodiments, the TIL obtained at 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 in the immunoglobulin, in the heavy chain of the immunoglobulin. In some embodiments, the diversity is in the immunoglobulin, in the immunoglobulin light chain. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is 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, the expression of TCRab (i.e., tcra/β) is 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 second amplification (e.g., according to step D of fig. 1) is performed in a closed system bioreactor. In some embodiments, the TIL amplification 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.

1. Feeder cells and antigen presenting cells

In one embodiment, the second amplification procedure described herein (e.g., including those such as described in step D of fig. 1 and those known as REP) requires an excess of feeder cells during the amplification of the REP TIL and/or during the second amplification. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units obtained from healthy blood donors. PBMCs were obtained using standard methods such as Ficoll-Paque gradient separation.

In general, allogeneic PBMCs are deactivated by irradiation or heat treatment, and as described in the examples are used in the REP procedure, which provides an exemplary protocol for assessing the inability of irradiated allogeneic PBMCs to replicate.

In some embodiments, PBMCs are considered replication-incompetent and accepted for the TIL expansion procedure described herein if the total number of surviving cells on day 14 is less than the number of initial surviving 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 accepted for the TIL expansion procedure described herein if the total number of surviving cells on days 7 and 14 of culture in the presence of OKT3 and IL-2 does not increase from the initial number of surviving 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 accepted for the TIL expansion procedure described herein if the total number of surviving cells on days 7 and 14 of culture in the presence of OKT3 and IL-2 does not increase from the initial number of surviving 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, the PBMC are cultured in the presence of 20 to 40ng/mL OKT3 antibody and 2000 to 4000IU/mL IL-2. In some embodiments, the PBMCs 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 one embodiment, the ratio of TIL to antigen presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In one embodiment, the proportion of TIL to antigen presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In one embodiment, the proportion of TIL to antigen presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.

In one embodiment, the second amplification procedure described herein requires about 2.5X10 ⁹ Pairs of individual feeder cells were approximately 100X 10 ⁶ Proportion of individual TILs. In another embodiment, the second amplification procedure described herein requires about 2.5X10 ⁹ Pairs of individual feeder cells were approximately 50X 10 ⁶ Proportion of individual TILs. In yet another embodiment, the second amplification procedure described herein requires about 2.5X10 ⁹ Pairs of individual feeder cells were approximately 25X 10 ⁶ And TIL.

In one embodiment, the second amplification procedure described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units obtained from healthy blood donors. PBMCs were obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting (aAPC) cells are used in place of PBMCs.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment for the TIL amplification procedures described herein, including the exemplary procedures described in the figures and examples.

In one embodiment, artificial antigen presenting cells are used in the second expansion to replace or in combination with PBMCs.

2. Cytokines and other additives

Alternatively, it is additionally possible to use a combination of cytokines for rapid amplification and or a second amplification of TIL, such as a combination of two or more of IL-2, IL-15 and IL-21 as described in U.S. patent application publication No. US 2017/0107490 A1, the disclosure of which is incorporated herein by reference in its entirety. 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, with the latter having particular utility in many embodiments. The use of a combination of cytokines is particularly advantageous for lymphocyte production, particularly T cells as described therein.

In one embodiment, step D may also comprise adding OKT-3 antibody or moromiab to the medium as described elsewhere herein. In one embodiment, step D may also include adding a 4-1BB agonist to the medium as described elsewhere herein. In one embodiment, step D may also comprise adding an OX-40 agonist to the medium as described elsewhere herein. Furthermore, additives may be used in the medium during step D, for example peroxisome proliferator activated receptor gamma coactivator I-alpha-agonists, including proliferator activated receptor (PPAR) -gamma agonists such as thiazolidinedione compounds, as described in U.S. patent application publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference in its entirety.

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 than four 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 of collecting TIL are well known in the art and any such known methods may be employed in the present process. In some embodiments, the TIL is collected using an automated system.

Cell collectors and/or cell handling systems are available from a number of sources including, for example, fresenius Kabi, tomtec Life Science, perkin Elmer, and Inotech Biosystems International, inc. Any cell-based collector may be used in the present method. 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 Fresenius Kabi). 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, such as 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 the need for agglomeration. 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 employed 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 the sterility and containment characteristics of the system. In some embodiments, a closed system as described in the examples is employed.

In some embodiments, TILs are collected according to the methods described in the examples. In some embodiments, TIL between day 1 and day 11 is collected using the methods described in the steps referred to herein (e.g., day 11 TIL collection in the examples). In some embodiments, TIL between day 12 and day 22 is collected using the methods described in the steps referred to 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 to a container (e.g., an infusion bag or sterile vial) for administration to a patient. In some embodiments, once a therapeutically sufficient amount of TIL is obtained using the amplification methods described above, they are transferred to a container for administration to a patient.

In one embodiment, the APC amplified TIL of the present disclosure is used as a pharmaceutical composition to be administered to a patient. In one embodiment, 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 as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

IV, 3 rd generation TIL manufacturing Process

Without being bound by any particular theory, it is believed that the initial first expansion (priming first expansion) that initiates activation of T cells as described by the methods of the invention, followed by the rapid second expansion that enhances activation of T cells, allows for the preparation of expanded T cells that retain a "younger" phenotype, thus it is expected that expanded T cells of the invention may exhibit higher cytotoxicity to cancer cells than T cells expanded by other methods. Specifically, it is believed that activation of T cells by exposure to an anti-CD 3 antibody (e.g., OKT-3), IL-2, and optionally Antigen Presenting Cells (APCs) as taught by the methods of the invention, and then enhanced by subsequent exposure to additional anti-CD 3 antibodies (e.g., OKT-3), IL-2, and APCs, limits or avoids maturation of T cells in culture, resulting in a population of T cells with less mature phenotypes that are less depleted by culture expansion and exhibit higher cytotoxicity to cancer cells. In some embodiments, the step of rapid second amplification is divided into the following steps to achieve a vertical scale up (scale up) of the culture scale: (a) Rapid second expansion is performed 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 to 4 days, followed by (b) transferring T cells in small-scale culture to a second vessel (e.g., G-REX 500MCS vessel) that is larger than the first vessel and culturing T cells from small-scale culture in a larger culture in the second vessel for a period of about 4 to 7 days. In some embodiments, the step of rapid amplification is divided into the following steps to achieve lateral scale up (scale out): (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 to 4 days, followed by (b) transferring and partitioning 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 vessels of equal size to the first vessel, wherein in each second vessel, the portion of T cells from the first small-scale culture transferred to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Performing 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 to 4 days, followed by (b) transferring and partitioning 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 vessels (e.g., g., G-REX 500MCS vessels) of larger size than the first vessel, wherein in each second vessel, the portion of T cells from the small-scale culture transferred to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Rapid second expansion is performed 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, followed by (b) transferring and dispensing T cells 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, wherein in each second vessel, the T cell fraction from the small-scale culture transferred to the second vessel is cultured in the larger-scale culture for a period of about 5 days.

In some embodiments, the rapid second expansion occurs after T cell activation caused by the initial first expansion begins to decrease, slow, decline or regress.

In some embodiments, the rapid second expansion occurs after T cell activation by the initial first expansion has been reduced by exactly 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 by the initial first expansion has been reduced by a percentage in the range of exactly or about 1% to 100%.

In some embodiments, the rapid second expansion occurs after T cell activation by the initial first expansion has been reduced by a percentage in the range of exactly or about 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 caused by the initial first expansion has been reduced by at least exactly 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 caused by the initial 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 caused by the initial 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 over 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 over a period of up to just 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 over 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 over a period of up to just or about 11 days.

In some embodiments, the rapid second expansion of T cells is performed over a period of up to just 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 some embodiments, the rapid second expansion of T cells is performed over 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 over a period of time of from about 1 day to about 7 days, and the rapid second expansion of T cells is performed over a period of time of from about 1 day to about 11 days.

In some embodiments, the initial first expansion of T cells is performed over a period of up to just or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells is performed over a period of up to just or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or about 11 days.

In some embodiments, the initial first expansion of T cells is performed over a period of time of from about 1 day to about 8 days, and the rapid second expansion of T cells is performed over a period of time of from about 1 day to about 9 days.

In some embodiments, the initial first expansion of T cells is performed over a period of 8 days and the rapid second expansion of T cells is performed over a period of 9 days.

In some embodiments, the initial first expansion of T cells is performed over a period of time of from about 1 day to about 7 days, and the rapid second expansion of T cells is performed over a period of time of from about 1 day to about 9 days.

In some embodiments, the initial first expansion of T cells is performed over a period of 7 days and the rapid second expansion of T cells is performed over a period of 9 days.

In some embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL).

In some embodiments, the T cell is a bone Marrow Infiltrating Lymphocyte (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 a patient with cancer.

In some embodiments, the T cells are MILs obtained from bone marrow of a hematological malignancy patient.

In some embodiments, the T cells are PBLs obtained from Peripheral Blood Mononuclear Cells (PBMCs) 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 the following: 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 suffers from hematological malignancies.

In certain aspects of the invention, immune effector cells, e.g., T cells, may be obtained from a unit of blood collected from a subject, isolated using any number of techniques known to those skilled in the art, such as FICOLL. In a preferred aspect, the cells in the circulating blood from the subject are obtained by blood cell separation. The blood cell separation product typically contains lymphocytes, including T cells, monocytes, granulosa cells, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by blood cell separation may be washed to remove plasma fractions and optionally the cells placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with Phosphate Buffered Saline (PBS). In an alternative embodiment, the wash solution lacks calcium, and may lack magnesium or may lack many if not all divalent cations. In one aspect, T cells are isolated from peripheral blood lymphocytes, which can be elutriated, for example, by centrifugation, by a PERCOLL gradient, or by countercurrent centrifugation, to lyse red blood cells and deplete monocytes.

In some embodiments, the T cells are PBLs isolated from whole donor blood or lymphocyte-enriched blood cell isolation products. 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 kidney Cell cancer. In some embodiments, the cancer is selected from the following: 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 suffers from hematological malignancies. In some embodiments, the PBLs are isolated from whole blood or lymphocyte-enriched blood cell isolation products using positive or negative selection methods, i.e., the PBLs are removed using a T cell phenotype marker such as cd3+cd45+, or non-T cell phenotype cells are removed while the PBLs remain. 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 be performed according to the initial first amplification step in any of the methods described herein by isolating an appropriate amount of isolated PBLs (in some embodiments, about 1 x 10 ⁷ PBLs) were inoculated into the initial first amplification culture.

An exemplary TIL process, referred to herein as process 3 (also referred to as generation 3), containing some of these features is depicted in fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C), and some of the advantages of this embodiment of the invention over generation 2 are depicted in fig. 1, 2, 30, and 31 (specifically, e.g., fig. 8B and/or fig. 8C). Two embodiments of process 3 are shown in fig. 1 and 30 (specifically, e.g., fig. 8B and/or fig. 8C). Generation 2 or generation 2A is also described in U.S. patent publication 2018/0280436, which is incorporated by reference in its entirety. The 3 rd generation procedure is also described in USSN 62/755,954 (filed on 5 th month of 2018) (116983-5045-PR).

As discussed and generally summarized herein, TILs are taken from patient samples and operated using the TIL amplification procedure described herein and referred to as generation 3 to amplify the amount thereof prior to implantation into a patient. In some embodiments, the TIL may optionally be genetically manipulated as discussed below. In some embodiments, the TIL may be cryopreserved prior to or after amplification. Once thawed, they may also be re-stimulated to increase their metabolism prior to infusion into a patient.

In some embodiments, as discussed in detail below and in the examples and figures, the initial first amplification (including the process referred to herein as rapid pre-amplification (pre-REP) and the process shown in fig. 8 (particularly, e.g., fig. 8B and/or fig. 8C) step B) 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 in fig. 1 (particularly, e.g., fig. 8B and/or fig. 8C) step D) is shortened to 1 to 9 days. In some embodiments, as discussed in detail below and in the examples and figures, the initial first amplification (including the process referred to herein as rapid pre-amplification (pre-REP) and the process shown in fig. 1 (particularly, e.g., fig. 8B and/or fig. 8C) step B) 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 in fig. 1 (particularly, e.g., fig. 8B and/or fig. 8C) step D) is shortened to 1 to 8 days. In some embodiments, as discussed in detail below and in the examples and figures, the initial first amplification (including the process referred to herein as rapid pre-amplification (pre-REP) and the process shown in fig. 1 (particularly, e.g., fig. 8B and/or fig. 8C) step B) 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 in fig. 1 (particularly, e.g., fig. 8B and/or fig. 8C) step D) is shortened to 1 to 9 days. In some embodiments, as discussed in detail below and in the examples and figures, the initial first amplification (including the process referred to herein as rapid pre-amplification (pre-REP) and the process shown in fig. 1 (particularly, e.g., fig. 1B and/or fig. 8C) step B) 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 in fig. 1 (particularly, e.g., fig. 8B and/or fig. 8C) step D) is 1 to 10 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 1 (e.g., fig. 8B and/or fig. 8C)) is shortened to 8 days and the rapid second amplification (e.g., the amplification described in step D of fig. 1 (e.g., fig. 8B and/or fig. 8C)) is shortened to 7 to 9 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 1 (e.g., fig. 8B and/or fig. 8C)) is 8 days and the rapid second amplification (e.g., the amplification described in step D of fig. 1 (e.g., fig. 8B and/or fig. 8C)) is 8 to 9 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 1 (e.g., fig. 8B and/or fig. 8C)) is shortened to 7 days and the rapid second amplification (e.g., the amplification described in step D of fig. 1 (e.g., fig. 8B and/or fig. 8C)) is shortened to 7 to 8 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is shortened to 8 days and the rapid second amplification (e.g., the amplification described in step D of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is shortened to 8 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 8 days and the rapid second amplification (e.g., the amplification described in step D of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 9 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 8 days and the rapid second amplification (e.g., the amplification described in step D of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 10 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 7 days and the rapid second amplification (e.g., the amplification described in step D of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 7 to 10 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 7 days and the rapid second amplification (e.g., the amplification described in step D of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 8 to 10 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 7 days and the rapid second amplification (e.g., the amplification described in step D of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is 9 to 10 days. In some embodiments, the initial first amplification (e.g., the amplification described in step B of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is shortened to 7 days and the rapid second amplification (e.g., the amplification described in step D of fig. 8 (e.g., fig. 8B and/or fig. 8C)) is shortened to 7 to 9 days. In some embodiments, as discussed in detail below and in the examples and figures, the combination of the initial first amplification and the rapid second amplification (e.g., the amplification described in step B and step D of fig. 1 (and in particular, e.g., fig. 1B and/or fig. 8C)) is 14 to 16 days. It is specifically contemplated that certain embodiments of the invention 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 TILs activated in the initial first expansion step as described above are a first population of TILs, i.e., a population of primary cells.

The following "step" code A, B, C, etc., refers to the non-limiting example of fig. 8 (specifically, e.g., fig. 8B and/or 8C) and to certain non-limiting embodiments described herein. The following and the sequence of steps in fig. 8 (specifically, e.g., fig. 8B and/or 8C) are exemplary and any combination or sequence of steps as well as additional steps, repetition steps and/or omission of steps are contemplated in the present application and methods disclosed herein.

A. Step A: obtaining a tumor sample of a patient

Generally, TIL is initially obtained from a patient tumor sample ("primary TIL") or circulating lymphocytes (e.g., peripheral blood lymphocytes, including peripheral blood lymphocytes having TIL-like characteristics), followed by expansion into a larger population for further manipulation as described herein, optionally cryopreserved and optionally assessed for phenotypic 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 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 hematological malignancy. The solid tumor may be any cancer species 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)), glioblastoma (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, useful TILs are obtained from malignant melanoma tumors, as reports indicate that these tumors have a particularly high amount of TILs.

Once obtained, the tumor sample is typically fragmented into pieces ranging from 1 to about 8mm using a sharp instrument ³ Small pieces of about 2 to 3mm ³ Is especially useful. TIL was cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., losv-pak souvenir 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 obtained by placing the tumor in an enzyme medium and mechanically dissociating the tumor for about 1 minute, followed by 5% CO at 37 ℃ ₂ After 30 minutes of incubation, the mechanical dissociation and incubation cycle was repeated under the conditions described above until only small tissue pieces were present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using FICOLL branched hydrophilic polysaccharides 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 in its entirety. 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 indicated above, in some embodiments, the TIL is derived from a solid tumor. In some embodiments, the solid tumor is not fractured. In some embodiments, the solid tumor is not fragmented and the enzyme digestion is performed with a 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 treated with 5% CO at 37 ℃ in an enzyme mixture comprising collagenase, dnase, and hyaluronidase ₂ Digestion was performed for 1 to 2 hours. In some embodiments, the tumor is treated with 5% CO at 37 ℃ in an enzyme mixture comprising collagenase, dnase, and hyaluronidase ₂ Digestion under rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is at 37 ℃, 5% CO ₂ Digestion was carried out overnight under constant rotation. In some embodiments, the whole tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In general, cell suspensions obtained from tumors are referred to as "primary cell populations" or "freshly obtained" or "freshly isolated" cell populations. In certain embodiments, the population of freshly obtained TIL cells is exposed to a cell culture medium comprising antigen presenting cells, IL-12 and OKT-3.

In some embodiments, when the tumor is a solid tumor, the tumor is physically broken after obtaining a tumor sample in step a, e.g., as provided in fig. 8 (particularly, e.g., fig. 8B and/or fig. 8C). In some embodiments, the disruption occurs prior to cryopreservation. In some embodiments, the disruption occurs after cryopreservation. In some embodiments, the disruption occurs after the tumor is obtained and no cryopreservation is present. In some embodiments, the disruption step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, or more than 40 fragments or pieces are placed into each container for initial first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed into each container for initial first amplification. In some embodiments, the tumor is fragmented and 40 fragments or patches are placed into each container for initial first amplification. In some embodiments, the plurality of fragments comprises from about 4 to about 50 fragments, each fragment having about 27mm ³ Is a volume of (c). In some embodiments, the plurality of segments comprises about 30 to about 60 segments, with a total volume of about 1300mm ³ Up to about 1500mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, the total volume of which is 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 Between them. In some embodiments, the tumor fragments are between about 1mm ³ And 8mm ³ Between them. In some embodiments, the tumor fragments are about 1mm ³ . In some embodiments, the tumor fragments are about 2mm ³ . In some embodiments of the present invention, in some embodiments,tumor fragments of 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 1-4mm by 1-4mm. 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 fragmented to minimize the amount of bleeding, necrosis, and/or adipose tissue on each patch. In some embodiments, the tumor is a tumor that is fractured to minimize the amount of bleeding tissue on each piece. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each patch. In some embodiments, the tumor is fragmented to minimize the amount of adipose tissue on each piece. In certain embodiments, the tumor-disrupting step is an in vitro or ex vivo method.

In some embodiments, tumor morcellation is performed to maintain tumor internal structure. In some embodiments, the performing of tumor morcellation does not include performing a sawing action using a scalpel. In some embodiments, the TIL is obtained from tumor digests. In some embodiments, tumor digests are produced by incubation in an enzyme medium such as, but not limited to, RPMI1640, 2mM Glutamax, 10mg/mL gentamicin, 30U/mL DNase, and 1.0mg/mL collagenase, followed by mechanical dissociation (GentleMACS, miltenyi Biotec, ornith, calif.). After placing the tumor in the enzyme medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be treated at 37℃with 5% CO ₂ For 30 minutes, followed by mechanical disruption again for about 1 minute. At 37℃at 5% CO ₂ After an additional 30 minutes incubation, tumors can be removedThe third time about 1 minute of mechanical disruption. In some embodiments, if a large piece of tissue is still present after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, whether or not at 5% CO at 37 ℃ anymore ₂ For 30 minutes. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using Ficoll to remove these cells.

In some embodiments, the cell suspension prior to the initial 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) and stored frozen prior to entering the expansion described in step B, which is described in further detail below and illustrated in fig. 8 (particularly, e.g., fig. 8B).

1. Boll/small biopsy derived TIL

In some embodiments, the TIL is initially obtained from a patient tumor sample ("primary TIL") by a core biopsy or similar procedure and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved and optionally assessed for phenotypic and metabolic parameters.

In some embodiments, patient tumor samples may be obtained using methods known in the art, typically by small biopsy, core biopsy, needle 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 hematological 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 coarse needle section 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 metastatic and the primary foci have been effectively treated/removed in the past, it may be possible to remove one metastatic foci as desired. In some embodiments, the least invasive way is to remove available skin lesions or cervical or axillary lymph nodes. 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, lung or liver metastatic lesions or intra-abdominal or thoracic lymph nodes or small biopsies thereof may be employed.

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 (punch biopsy). In some embodiments, the perforated biopsy is obtained by pressing a circular blade into the skin. In some embodiments, the perforated biopsy is obtained by pressing a circular blade into the skin surrounding the suspicious mole. In some embodiments, 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 growth is 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 growth.

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 black moles are very large.

In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Typically, bronchoscopy is performed under patient anesthesia with a small tool 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, tumors or growths are not accessible by bronchoscopy, and percutaneous aspiration of biopsy may be used. Typically, percutaneous aspiration of biopsy is also under patient anesthesia, with a needle inserted directly through the skin into the suspicious site to remove a small sample of tissue. In some embodiments, aspiration of a biopsy of a body tissue via 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 via endoscopic ultrasound (e.g., endoscopic oral placement into the esophagus with a light). 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, sample collection may not require hospitalization if the abnormal area is easily accessible. In some embodiments, if the tumor is deeper within 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 very fine needles attached to a syringe are used to withdraw (aspirate) cells from a tumor or tumor mass. 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 site 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 slice a small portion of the cervical surface of a living tissue. 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, wherein an outpatient procedure may be required to remove a larger piece of cervical tissue. In some embodiments, a cone-shaped biopsy may be used as an initial treatment in addition to helping confirm the diagnosis.

The term "solid tumor" refers to an abnormal mass of tissue, typically free of cysts or areas of fluid. Solid tumors may be benign or malignant. The term "solid tumor cancer" refers to malignant, neoplastic or cancerous solid tumors. Solid tumor cancers include lung cancer. 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 as a Fine Needle Aspirate (FNA), a core 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 core biopsy and/or small biopsy samples, the sample is first placed in G-Rex 10. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more than 10 core biopsy and/or small biopsy samples, the sample is first placed in G-Rex 100. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more than 10 core biopsy and/or small biopsy samples, the sample is first placed in G-Rex 500.

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 certain instances, NSCLC patients have been previously surgically treated.

TIL as described herein may be obtained from FNA samples. In certain instances, FNA samples are obtained or isolated from patients using fine gauge needles ranging from 18 gauge needles to 25 gauge needles. The gauge needle may be 18 gauge, 19 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge, 24 gauge, or 25 gauge. In some embodiments, a FNA sample from a 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 than 950,000 TILs.

In some embodiments, the TILs described herein are obtained from a core biopsy sample. In certain instances, core biopsy samples are obtained or isolated from a patient using surgical or medical needles ranging from 11 gauge needles to 16 gauge needles. The needle may be 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge, or 16 gauge. In some embodiments, a core 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 than 950,000 TILs.

In some embodiments, the TIL is not obtained from tumor digests. In some embodiments, the solid tumor coarse needle section is not broken.

In some embodiments, the TIL is obtained from tumor digests. In some embodiments, 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, miltenyi Biotec, ornith, calif.). After placing the tumor in the enzyme medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be treated at 37℃with 5% CO ₂ For 30 minutes, followed by mechanical disruption again for about 1 minute. At 37℃at 5% CO ₂ After an additional 30 minutes of incubation, the tumor may be mechanically destroyed a third time for about 1 minute. In some embodiments, if a large piece of tissue is still present after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, whether or not at 5% CO at 37 ℃ anymore ₂ For 30 minutes. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using Ficoll to remove these cells.

In some embodiments, obtaining the first TIL population comprises a multi-foci sampling method.

The tumor dissociating enzyme mixture may include more than one dissociating (digesting) enzyme, such as, but not limited to collagenase (including collagenase of any blend or type), accutase ^TM 、Accumax ^TM Hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (chain protease), deoxyribonuclease I (dnase), trypsin inhibitor, any other dissociating or proteolytic enzyme Any combination thereof.

In some embodiments, the dissociating enzyme is reconstituted from a freeze-dried enzyme. In some embodiments, the lyophilized enzyme is reconstituted with an amount of a sterile buffer such as Hank's Balanced Salt Solution (HBSS).

In some embodiments, the stock solution of enzyme may be altered to verify the concentration of the freeze-dried stock solution and modify the final amount of enzyme added to the digestion mixture accordingly.

In some embodiments, the enzyme mixture comprises about 10.2-ul of neutral protease (0.36 DMC U/mL), 21.3-ul of collagenase (1.2 PZ/mL), and 250-ul of DNase I (200U/mL) in about 4.7mL of sterile HBSS.

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 pleural effusion derived sample. 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 No. US 2014/0295426, 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, such as 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 TIL-containing slurries, including, for example, ascites in the abdomen or pancreatic cyst fluid. Ascites and pleural fluids involve very similar chemical systems; both the abdomen and the lungs have mesothelial cell lines and in malignant disease in the same situation form fluids in the pleural and abdominal spaces, such fluids containing TIL in some embodiments. In some embodiments, where the present disclosure exemplifies pleural fluid, the same procedure may be performed using ascites or other cyst fluid containing TIL to achieve similar results.

In some embodiments, the pleural fluid is untreated, straightSuch as in a form that is removed from the patient. 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 step In a test tube (Veridex). In some embodiments, samples are placed into CellSave tubes immediately after collection from the patient to avoid a decrease in the number of surviving TILs. If left in untreated pleural fluid even at 4 ℃, the number of surviving TILs can be reduced to a significant extent within 24 hours. In some embodiments, the sample is placed into an appropriate collection tube within 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 into an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4 ℃.

In some embodiments, a pleural fluid sample (including, for example, untreated pleural fluid), diluted pleural fluid, or a mass of resuspended cells is contacted with a lysing agent that differentially lyses the non-nucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in situations where the pleural fluid contains a large number of RBCs. Suitable lysing reagents include a single lysing reagent or lysing reagent and a quenching reagent or lysing reagent, quenching reagent and a solidAnd (5) determining a reagent. Suitable cleavage systems are commercially available and include BD Pharm Lyse ^TM System (Becton Dickenson). Other cleavage systems include Versalysie ^TM System, FACSly ^TM System (Becton Dickenson), immunoprep ^TM The system or the Erythrolyse II system (Beckman Coulter, inc.) or the ammonium chloride system. In some embodiments, the lysing agent may vary with the phenotypic properties of TIL and TIL in the primary requirement for effective lysis of red blood cells and retention of pleural fluid. In addition to employing a single lysing reagent, the lysing systems useful in the methods described herein may include a second reagent, such as a reagent that quenches or blocks the effect of the lysing reagent during the remaining steps of the method, such as stabilysine ^TM Reagents (Beckman Coulter, inc.). Depending on the choice of cleavage reagent or the preferred execution of the method, well known immobilization reagents may also be used.

3. Method for amplifying Peripheral Blood Lymphocytes (PBLs) derived from peripheral blood

PBL method 1. In an embodiment of the invention, the PBLs are amplified using the procedure described herein. In an embodiment of the invention, the method comprises obtaining a PBMC sample from whole blood. In one embodiment, the method comprises enriching T cells by using a negative selection non-cd19+ fraction to isolate pure T cells from PBMCs. In one embodiment, the method comprises enriching T cells by using a magnetic bead-based negative selection non-cd19+ fraction to isolate pure T cells from PBMCs.

In one embodiment of the invention, the PBL method 1 proceeds as follows: on day 0, the cryopreserved PBMC samples were thawed and PBMCs were counted. T cells were isolated using a human pan T cell isolation kit and LS column (Miltenyi Biotec).

PBL method 2. In one embodiment of the invention, PBL is amplified using PBL method 2, which includes obtaining a PBMC sample from whole blood. By incubation at 37 ℃PBMCs were incubated for at least three hours and then non-adherent cells were isolated to enrich T cells from PBMCs.

In one embodiment 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 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 one embodiment of the invention, PBL is amplified using PBL method 3, which includes obtaining a PBMC sample 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 one embodiment of the present invention, PBL method 3 proceeds as follows: on day 0, cryopreserved PBMCs derived from peripheral blood were thawed and counted. Cd19+ B cells were sorted using the CD19Multisort human kit (Miltenyi Biotec). In the non-cd19+ cell fraction, T cells were purified using a human pan T cell isolation kit and LS column (Miltenyi Biotec).

In one embodiment, the PBMCs are isolated from a whole blood sample. In one embodiment, PBMC samples are used as starting material for the amplification of PBLs. In one embodiment, the sample is cryopreserved prior to the amplification process. In another embodiment, fresh samples are used as starting materials for the amplification of PBLs. In embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In one embodiment, T cells are isolated using a human pan T cell isolation kit and LS column. In embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art (e.g., CD19 negative selection).

In an embodiment of the invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify non-adherent cells. In an embodiment of the invention, the incubation time is about 3 hours. In an embodiment of the invention, the temperature is about 37 ℃. Non-adherent cells are then expanded using the procedure described above.

In some embodiments, the PBMC sample is derived from a subject or patient that has been optionally pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is derived 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 ITK inhibitor 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 another embodiment, the PBMCs are derived from a patient currently undergoing ITK inhibitor regimen such as ibrutinib treatment.

In some embodiments, the PBMC samples are derived from subjects or patients that have been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and that are refractory to treatment with a kinase inhibitor or an ITK inhibitor, such as 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, 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 ITK inhibitor but is no longer treated with the kinase inhibitor or ITK inhibitor and is not 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 more. In another embodiment, the PBMCs are derived from a patient previously exposed to an ITK inhibitor but who has not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In an embodiment of the invention, on day 0, the cells are cd19+ selected and sorted accordingly. In an embodiment of the invention, the selection is performed using antibody binding beads. In an embodiment of the invention, the pure T cells are isolated from PBMCs on day 0.

In one embodiment of the invention, 10 to 15mL of white blood cell layer will yield about 5X 10 for a patient not previously treated with ibrutinib or other ITK inhibitor ⁹ Individual PBMCs, which in turn will produce about 5.5 x 10 ⁷ And PBLs.

In one embodiment of the invention, the amplification process will result in about 20X 10 for patients who have been treated with ibrutinib or other ITK inhibitors ⁹ And PBLs. In one embodiment of the invention 40.3X10 ⁶ The individual PBMC will produce approximately 4.7X10 ⁵ And PBLs.

In any of the foregoing embodiments, the PBMCs may be derived from a whole blood sample, by blood cell separation, from a white blood cell layer, or from any other method known in the art for obtaining PBMCs.

In any of the foregoing embodiments, the PBL can be genetically modified to express a CCR as described herein. In some embodiments, the PBL is prepared using the method described in U.S. patent application publication No. US 2020/0347350 A1, the disclosure of which is incorporated herein by reference in its entirety.

4. Method for amplifying bone Marrow Infiltrating Lymphocytes (MILs) from bone marrow derived PBMCs

MIL method 3. In one embodiment of the invention, the method comprises obtaining a PBMC sample from bone marrow. On day 0, PBMC were selected and sorted by CD3+/CD33+/CD20+/CD14+, non-CD3+/CD33+/CD20+/CD14+ cell fractions were sonicated and a portion of the sonicated cell fraction was added back to the selected cell fraction.

In one embodiment of the invention, MILs method 3 proceeds as follows: on day 0, the cryopreserved samples of PBMCs were thawed and PBMCs 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 (or MILs fraction) (cd3+cd33+cd20+cd14+) and AML blast fraction (non-cd3+cd33+cd20+cd14+).

In one embodiment of the invention, the PBMCs are obtained from bone marrow. In one embodiment, the PBMCs are obtained from bone marrow by blood cell separation, aspiration, needle aspiration of living tissue sections, or other similar means known in the art. In one embodiment, the PBMCs are fresh. In another embodiment, the PBMCs are cryopreserved.

In an embodiment of the invention, MILs are amplified from 10 to 50mL of bone marrow aspirate. In an embodiment of the invention, 10mL of bone marrow aspirate is obtained from a patient. In another embodiment, 20mL of bone marrow aspirate is obtained from a patient. In another embodiment, 30mL of bone marrow aspirate is obtained from a patient. In another embodiment, 40mL of bone marrow aspirate is obtained from a patient. In another embodiment, 50mL of bone marrow aspirate is obtained from a patient.

In an embodiment of the invention, the number of PBMC generated from about 10 to 50mL of bone marrow aspirate is about 5X 10 ⁷ Up to about 10X 10 ⁷ PBMCs were used. In another embodiment, the number of PMBC produced is about 7×10 ⁷ PBMCs were used.

In one embodiment of the invention, about 5X 10 ⁷ Up to about 10X 10 ⁷ The individual PBMC produced about 0.5X10 ⁶ To about 1.5X10 ⁶ And MIL. In one embodiment of the invention, about 1X 10 is produced ⁶ And MIL.

In one embodiment of the invention, the bone marrow aspirate is derived from 12X 10 ⁶ The individual PBMC produced about 1.4X10 ⁵ And MIL.

In any of the foregoing embodiments, the PBMCs may be derived from a whole blood sample, bone marrow, isolated by blood cells, from a white blood cell layer, or from any other method known in the art for obtaining PBMCs.

In any of the foregoing embodiments, MILs can be genetically modified to express CCR as described herein. In some embodiments, MILs are prepared using the methods described in U.S. patent application publication No. US 2020/0347350 A1, the disclosure of which is incorporated herein by reference in its entirety.

B. And (B) step (B): initial first amplification

In some embodiments, the present methods provide a younger TIL that may provide additional therapeutic benefits compared to an older TIL (i.e., a TIL that has been replicated more times further prior to administration to a subject/patient). Features of young TILs have been described in the literature, for example Donia et al, scand.j. Immunol.2012,75,157-167; dudley et al, clin.cancer Res.2010,16,6122-6131; huang et al, J.Immunother.2005,28,258-267; besser et al, clin.cancer Res.2013,19, OF1-OF9; besser et al, J.Immunother.2009,32,415-423; robbins et al, J.Immunol.2004,173,7125-7130; shen et al, j.immunother, 2007,30,123-129; zhou et al, J.Immunother.2005,28,53-62; and Tran et al, j.immunother, 2008,31,742-751, each of which is incorporated herein by reference in its entirety.

After the tumor fragments and/or tumor fragments are segmented or digested, for example, such as described in step a of fig. 1 (and in particular, e.g., fig. 1B and/or fig. 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 along with tumor digests and/or tumor fragments (e.g., on day 0). In some embodiments, tumor digests and/or tumor fragments are incubated in the container with up to 60 fragments per container and 6000IU/mL of IL-2. In some embodiments, this primary cell population is cultured for a period of days (typically 1 to 8 days), resulting in a typical cell population of about 1 x 10 ⁸ A host population of individual host TIL cells. In some embodiments, this primary cell population is cultured for a period of days (typically 1 to 7 days), resulting in a typical cell population of about 1 x 10 ⁸ A host population of individual host TIL cells. In some embodiments, the initial first amplification occurs for a period of 1 to 8 days, resulting in a typical of about 1 x 10 ⁸ A host population of individual host TIL cells. In some embodiments, the initial first amplification occurs for a period of 1 to 7 days, resulting in a typical of about 1 x 10 ⁸ A host population of individual host TIL cells. In some embodiments, this initial first amplification occurs over a period of 5 to 8 days, resulting in a typical of about 1×10 ⁸ A host population of individual host TIL cells. In some embodiments, this initial first amplification occurs for a period of 5 to 7 days, resulting in a typical of about 1×10 ⁸ A host population of individual host TIL cells. In some embodiments, this initial first amplification occurs over a period of about 6 to 8 days, resulting in a typical of about 1×10 ⁸ A host population of individual host TIL cells. In some embodiments, this initialThe first amplification occurs for a period of about 6 to 7 days, resulting in a typical of about 1×10 ⁸ A host population of individual host TIL cells. In some embodiments, this initial first amplification occurs over a period of about 7 to 8 days, resulting in a typical of about 1×10 ⁸ A host population of individual host TIL cells. In some embodiments, this initial first amplification occurs over a period of about 7 days, resulting in a typical of about 1×10 ⁸ A host population of individual host TIL cells. In some embodiments, this initial first amplification occurs over a period of about 8 days, resulting in a typical of about 1×10 ⁸ A host population of individual host TIL cells.

In a preferred embodiment, the amplification of TIL may be performed using an initial first amplification step as described below and herein (e.g., those described in fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C) step B, which may include a process called prep or initial REP and which contains feeder cells from day 0 and/or from the beginning of culture), followed by a rapid 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 restimulation REP step) as described below and herein. TIL obtained from this process can optionally be identified as described herein with phenotypic characteristics and metabolic parameters. In some embodiments, the tumor fragments are between about 1mm ³ And 10mm ³ Between them.

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 a GREX100MCS flask. In some embodiments, less than or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each vessel contains less than or equal to 500mL of medium per vessel. In some embodiments, the culturingThe 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 per vessel ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium comprises OKT-3. In some embodiments, the medium comprises 30ng/mL OKT-3 per container. In some embodiments, the container is a GREX100MCS flask. In some embodiments, the medium comprises 6000IU/mL IL-2, 30ng OKT-3, and 2.5X10 ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium contains 6000IU/mL IL-2, 30ng/mL OKT-3, and 2.5X10 per container ⁸ Individual antigen presenting feeder cells.

After preparation of tumor fragments, the resulting cells (i.e., as fragments of the primary cell population) were 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 allowed TIL initiation and accelerated growth from day 0 initiation of culture. In some embodiments, tumor digests and/or tumor fragments are incubated with 6000IU/mL IL-2, as well as 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 typical cell population of about 1X 10 ⁸ A host population of individual host TIL cells. In some embodiments, the growth medium during the initial first expansion 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 typical cell population of about 1 x 10 ⁸ A host population of individual host TIL cells. In some embodiments, the growth medium during the initial first expansion 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 20 to 30X 10 ⁶ IU/mg specific activity. In some embodiments, the 1mg vial of IL-2 stock solution has a 20X 10 ⁶ IU/mg specific activity. In some embodiments, IL-2 stock solution 1mgThe vials had 25 x 10 ⁶ IU/mg specific activity. In some embodiments, the 1mg vial of IL-2 stock solution has a 30X 10 ⁶ IU/mg specific activity. In some embodiments, the IL-2 stock solution has a length of 4 to 8X10 ⁶ The final concentration of IU/mg IL-2. In some embodiments, the IL-2 stock solution has a length of 5 to 7X 10 ⁶ The final concentration of IU/mg IL-2. In some embodiments, the IL-2 stock solution has a 6X 10 ⁶ The final concentration of 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 of IL-2, about 9,000IU/mL of IL-2, about 8,000IU/mL of IL-2, about 7,000IU/mL of IL-2, about 6000IU/mL of IL-2, or about 5,000IU/mL of IL-2. In some embodiments, the initial first amplification medium comprises from about 9,000IU/mL IL-2 to about 5,000IU/mL IL-2. In some embodiments, the initial first amplification medium comprises from about 8,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the initial first amplification medium comprises from about 7,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the initial first amplification medium comprises about 6,000IU/mL IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the initial first expanded cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, the initial first expanded cell culture medium further comprises IL-2. In a preferred embodiment, the initial first expanded cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, the initial 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 of IL-2. In one embodiment, the initial 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 of 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 initial first amplification medium comprises from about 500IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the initial first amplification medium comprises from about 400IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the initial first amplification medium comprises from 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 initial first expanded cell culture medium comprises about 180IU/mL IL-15. In one embodiment, the initial first expanded cell culture medium further comprises IL-15. In a preferred embodiment, the initial first expanded cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the initial first amplification medium comprises about 20IU/mL of IL-21, about 15IU/mL of IL-21, about 12IU/mL of IL-21, about 10IU/mL of IL-21, about 5IU/mL of IL-21, about 4IU/mL of IL-21, about 3IU/mL of IL-21, about 2IU/mL of IL-21, about 1IU/mL of IL-21, or about 0.5IU/mL of IL-21. In some embodiments, the initial first amplification medium comprises from about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the initial first amplification medium comprises from about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the initial first amplification medium comprises from about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the initial first amplification medium comprises from about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the initial 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 initial first expanded cell culture medium comprises about 1IU/mL IL-21. In some embodiments, the initial first expanded cell culture medium comprises about 0.5IU/mL IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the initial first expanded cell culture medium comprises about 1IU/mL IL-21.

In one embodiment, the initial first expanded cell culture medium comprises OKT-3 antibodies. In some embodiments, the initial first expanded cell culture medium comprises about 30ng/mL OKT-3 antibody. In one embodiment, the initial 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. Mu.g/mL OKT-3 antibody. In one embodiment, 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 one embodiment, the cell culture medium comprises between 15ng/mL and 30ng/mL OKT-3 antibody. In one embodiment, the cell culture medium comprises 30ng/mL OKT-3 antibody. In some embodiments, the OKT-3 antibody is Moromolizumab.

In some embodiments, the initial first expanded 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, and 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 initial first expanded cell culture medium 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 one or more TNFRSF agonists, one or more TNFRSF agonists comprising a 4-1BB agonist. In some embodiments, the initial first expanded cell culture medium 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 one or more TNFRSF agonists, one or more TNFRSF agonists comprising a 4-1BB agonist.

In some embodiments, the initial 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, CM is CM1 as described in the examples. In some embodiments, the initial first expansion occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial first expansion medium or initial 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 medium used in the amplification process disclosed herein is serum-free medium or defined medium. In some embodiments, the serum-free or defined medium comprises basal cell culture medium, serum supplements and/or serum substitutes. In some embodiments, serum-free or defined media is used to prevent and/or reduce experimental variation due in part to batch variation of serum-containing media.

In some embodiments, the serum-free or defined medium comprises basal cell culture medium, serum supplements and/or serum substitutes. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS ^TM OpTmizer ^TM T cell expansion basal medium, CTS ^TM OpTmizer ^TM T cell expansion SFM, CTS ^TM AIM-V Medium, CTS ^TM AIM-V SFM、LymphoONE ^TM T cell expansion Xeno-free medium, dulbecco's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential Medium (aMEM), glasgow minimal essential Medium (G-MEM), RPMI growth Medium and Iscove's modified Dulbecco ' sA culture medium.

In some embodiments, the serum supplement or serum replacement includes, but is not limited to, one or more CTS ^TM Optmizer T cell expansion 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 medium is determined to comprise 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 Ag containing trace elements ⁺ 、Al ³⁺ 、Ba ²⁺ 、Cd ²⁺ 、Co ²⁺ 、Cr ³⁺ 、Ge ⁴⁺ 、Se ⁴⁺ 、Br、T、Mn ²⁺ 、P、Si ⁴⁺ 、V ⁵⁺ 、Mo ⁶⁺ 、Ni ²⁺ 、Rb ⁺ 、Sn ²⁺ And Zr (Zr) ⁴⁺ Is a compound of (a). In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS ^TM OpTmizer ^TM The T cell immune cell serum replacement is used with conventional growth media including, but not limited to CTS ^TM OpTmizer ^TM T cell expansion basal medium, CTS ^TM OpTmizer ^TM T cell expansion SFM, CTS ^TM AIM-V medium, CST ^TM AIM-VSFM、LymphoONE ^TM T cell expansion Xeno-free medium, dulbecco's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential Medium (aMEM), glasgow minimal essential Medium (G-MEM), RPMI growth Medium and Iscove's modified Dulbecco ' sA culture medium.

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% of the total serum-free or defined medium volume. 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 ^TM OpTmizer ^TM T cell expansion SFM (ThermoFisher Scientific). Any CTS ^TM OpTmizer ^TM Formulations are useful in the present invention. CTS (clear to send) ^TM OpTmizer ^TM T cell expansion SFM 1L CTS ^TM OpTmizer ^TM T cell expansion basal medium and 26mL CTS ^TM OpTmizer ^TM T cell expansion supplements are mixed together prior to use. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with approximately 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), final concentration of 2-mercaptoethanol in the medium was 55. Mu.M.

In some embodiments, the medium is determined to be CTS ^TM OpTmizer ^TM T cell expansion SFM (ThermoFisher Scientific). Any CTS ^TM OpTmizer ^TM Formulations are useful in the present invention. CTS (clear to send) ^TM OpTmizer ^TM T cell expansion SFM 1L CTS ^TM OpTmizer ^TM T cell expansion basal medium and 26mL CTS ^TM OpTmizer ^TM T cell expansion supplements are mixed together prior to use. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol and 2mM L-glutamine. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising from about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol, further comprising from about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol, further comprising about 3000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol, further comprising about 1000IU/mL to about 6000IU/mLIL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, further comprising about 6000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immunocyte Serum Replacement (SR) (ThermoFisher Scientific) and final concentration of 2-mercaptoethanol in the medium was 55. Mu.M.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., 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). 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 described in International PCT patent publication No. WO/1998/030679 (incorporated herein by reference in its entirety) may be used in the present invention. In this publication, 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. Serum-free eukaryotic cell culture medium supplements comprise or are 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 medium is determined to comprise 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 Ag containing trace elements ⁺ 、Al ³⁺ 、Ba ²⁺ 、Cd ² ⁺ 、Co ²⁺ 、Cr ³⁺ 、Ge ⁴⁺ 、Se ⁴⁺ 、Br、T、Mn ²⁺ 、P、Si ⁴⁺ 、V ⁵⁺ 、Mo ⁶⁺ 、Ni ²⁺ 、Rb ⁺ 、Sn ²⁺ And Zr (Zr) ⁴⁺ Is a compound of (a). In some embodiments, the basal cell culture mediumSelected from the following: dulbecco's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (. Alpha.MEM), glasgow minimal essential medium (G-MEM), RPMI growth medium, and Iscove's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the 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 transferrin is about 1 to 250mg/L, the concentration of transferrin is about 10 to 500mg/L, the concentration of albumin is about 1 to 50mg to 0.0001mg/L (e.g., 0.0001mg/L, 0.01 mg/L.) I) Is about 5000 to 50,000mg/L.

In some embodiments, the non-trace element fraction component of the defined medium is present in the concentration range listed in the column entitled "concentration range in 1X medium" in table 4 below. In other embodiments, the non-trace element fraction component of the defined medium is present at the final concentrations listed in the column entitled "preferred embodiment of Medium 1X" in Table 4 below. 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 the column entitled "preferred embodiments of supplement" in table 4 below.

Table 4: concentration of non-trace element fraction

In some embodiments, the osmolality of the culture medium is determined to be 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 about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100. Mu.M), 2-mercaptoethanol (final concentration about 100. Mu.M).

In some embodiments, the defined media described in Smith et al, clin. Fransl. Immunology,2015,4 (1), e31 (the disclosure of which is incorporated herein by reference in its entirety) may be used in the present invention. Briefly, RPMI or CTS ^TM OpTmizer ^TM Used as basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS ^TM Immune cell serum replacement.

In one embodiment, the cell culture medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell culture media can simplify the procedure required to expand cell numbers. In one embodiment, 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).

In some embodiments, the initial first amplification (including, for example, those described in step B of fig. 1 (and in particular, for example, fig. 1B and/or fig. 8C), which may include those sometimes referred to as prep or initial REP) is performed for 1 to 8 days, as discussed in the examples and figures. In some embodiments, the initial first amplification (including, for example, those described in step B of fig. 1 (and in particular, for example, fig. 1B and/or fig. 8C), which may include those sometimes referred to as prep or initial REP) process is 2 to 8 days. In some embodiments, the initial first amplification (including, for example, those described in step B of fig. 1 (and in particular, for example, fig. 1B and/or fig. 8C), which may include those sometimes referred to as prep or initial REP) process is 3 to 8 days. In some embodiments, the initial first amplification (including, for example, those described in step B of fig. 1 (and in particular, for example, fig. 8B and/or fig. 8C), which may include those sometimes referred to as pre-REP or initial REP) process is 4 to 8 days, as discussed in the examples and figures. In some embodiments, the initial first amplification (including, for example, those described in step B of fig. 8 (and in particular, for example, fig. 8B and/or fig. 8C), which may include those sometimes referred to as pre-REP or initial REP) process is 1 to 7 days, as discussed in the examples and figures. In some embodiments, the initial first amplification (including, for example, those described in step B of fig. 1 (and in particular, for example, fig. 1B and/or fig. 8C), which may include those sometimes referred to as prep or initial REP) process is 2 to 8 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is 2 to 7 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is 3 to 8 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is 3 to 7 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is 4 to 8 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is 4 to 7 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is from 5 to 8 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is from 5 to 7 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is from 6 to 8 days. In some embodiments, the initial first amplification (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 prep or initial REP) process is from 6 to 7 days. In some embodiments, the initial first amplification (including, for example, those provided in step B of fig. 1 (particularly, for example, fig. 8B and/or fig. 8C), which may include those sometimes referred to as prep or initial REP) process is 7 to 8 days. In some embodiments, the initial first amplification (including, for example, those provided 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) process is 8 days. In some embodiments, the initial first amplification (including, for example, those provided 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) process is 7 days.

In some embodiments, the initial first TIL amplification may be performed from 1 day to 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed for 1 day to 7 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed 2 days to 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed 2 days to 7 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed 3 days to 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed 3 days to 7 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed 4 days to 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed 4 days to 7 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed for 5 days to 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed for 5 days to 7 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed from 6 days to 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed from 6 days to 7 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed 7 to 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed for 8 days after the disruption occurs and/or after the initiation of the first initial amplification step. In some embodiments, the initial first TIL amplification may be performed for 7 days after the disruption occurs and/or after the initiation of the first initial amplification step.

In some embodiments, 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 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 used as the combination during the initial first amplification. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included during the initial first amplification, including, for example, during the step B process according to FIG. 8 (particularly, e.g., FIG. 8B and/or FIG. 8C) and as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is used as the combination during the initial 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. 8 (specifically, e.g., FIG. 8B and/or FIG. 8C), and as described herein.

In some embodiments, the initial first amplification (e.g., according to step B of fig. 8 (particularly, e.g., fig. 8B and/or fig. 8C)) 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 one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 1 (and in particular, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added during initial first amplification. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during days 4 to 8 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during days 4 to 7 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during days 5 to 8 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during days 5 to 7 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during days 6-8 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during days 6 to 7 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during day 7 or 8 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during day 7 of the initial first amplification period. In one embodiment, the initial first amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or fig. 8C) step B and those referred to as prep or initial REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the beginning of TIL amplification, but rather is added at any time during day 8 during initial first amplification.

In one embodiment, the initial first expansion procedure described herein (e.g., including those such as described in step B of fig. 8 (and in particular, 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 of and during initial first expansion of the TIL. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units obtained from allogeneic healthy blood donors. PBMCs were obtained using standard methods such as Ficoll-Paque gradient separation. In some embodiments, 2.5X10 are used during the initial first amplification ⁸ And (3) feeder cells. In some embodiments, 2.5X10 per container is used during the initial first amplification ⁸ And (3) feeder cells. In some embodiments, 2.5X10 per GREX-10 is used during the initial first amplification ⁸ And (3) feeder cells. In some embodiments, 2.5X10 per GREX-100 is used during the initial first amplification ⁸ And (3) feeder cells.

In general, allogeneic PBMCs are deactivated by irradiation or heat treatment, as described in the examples for the REP procedure, which provides an exemplary protocol for assessing the inability of irradiated allogeneic PBMCs to replicate.

In some embodiments, PBMCs are considered replication-incompetent and acceptable for the TIL expansion procedure described herein if the total number of surviving cells on day 14 is less than the number of initial surviving cells placed in culture on day 0 of 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 surviving cells on day 7 of culture in the presence of OKT3 and IL-2 is not increased compared to the number of initial surviving 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 the TIL expansion procedure described herein if the total number of surviving cells on day 7 of culture in the presence of OKT3 and IL-2 is not increased compared to the number of initial surviving 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, the PBMC are cultured in the presence of 20 to 40ng/mL OKT3 antibody and 2000 to 4000IU/mL IL-2. In some embodiments, the PBMCs 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 one embodiment, the initial first amplification procedure described herein requires about 2.5X10 ⁸ Pairs of individual feeder cells were approximately 100X 10 ⁶ Proportion of individual TILs. In another embodiment, the initial first amplification procedure described herein requires about 2.5X10 ⁸ Pairs of individual feeder cells were approximately 50X 10 ⁶ Proportion of individual TILs. In yet another embodiment, the initial first amplification described herein requires about 2.5X10 ⁸ Pairs of individual feeder cells were approximately 25X 10 ⁶ And TIL. In yet another embodiment, the initial first amplification described herein requires about 2.5X10 ⁸ And (3) feeder cells. In yet another embodiment, the initial first expansion requires 1/4, 1/3, 5/12 or 1/2 of the number of feeder cells for the rapid second expansion.

In some embodiments, the medium of the initial first amplification comprises IL-2. In some embodiments, the medium of the initial first amplification comprises 6000IU/mL of IL-2. In some embodiments, the initial first expanded medium comprises antigen presenting feeder cells. In some embodiments, the medium of the initial first amplification comprises 2.5X10 per vessel ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium of the initial first amplification comprises OKT-3. In some embodiments, the medium comprises 30ng OKT-3 per container. In some embodiments, the container is a GREX100MCS flask. In some embodiments, the medium comprises 6000IU/mL IL-2, 30ng/mL OKT-3, and 2.5X10 ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium contains 6000IU/mL IL-2, 30ng/mL OKT-3, and 2.5X10 per container ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium comprises every 2.5X10 per container ⁸ 500mL of medium and 15. Mu.g of OKT-3. In some embodiments, the medium comprises 500mL of medium and 15 μg of OKT-3 per container. In some embodiments, the container is a GREX100MCS flask. In some embodiments, the medium comprises 500mL of medium, 6000IU/mL of IL-2, 30ng/mL of OKT-3, and 2.5X10 ⁸ Individual antigen presenting feeder cells. In some embodimentsWherein the medium comprises 500mL of medium per container, 6000IU/mL of IL-2, 15. Mu.g of OKT-3 and 2.5X10 ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium comprises every 2.5X10 per container ⁸ 500mL of medium and 15. Mu.g of OKT-3.

In one embodiment, the initial first amplification procedure described herein requires an excess of feeder cells over TIL during the second amplification. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units obtained from allogeneic healthy blood donors. PBMCs were obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting (aAPC) cells are used in place of PBMCs.

Generally, allogeneic PBMCs are deactivated by irradiation or heat treatment and are used in the TIL amplification procedures described herein, including the exemplary procedures described in the figures and examples.

In one embodiment, artificial antigen presenting cells are used to replace or in combination with PBMCs in the initial first expansion.

2. Cytokines and other additives

Alternatively, it is additionally possible to use a combination of cytokines for initial first amplification of TIL, such as a combination of two or more of IL-2, IL-15 and IL-21 as described in U.S. patent application publication No. US 2017/0107490 A1, the disclosure of which is incorporated herein by reference in its entirety. 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 lymphocyte production, particularly the T cells described therein.

In one embodiment, step B may also comprise adding OKT-3 antibody or moromiab to the medium as described elsewhere herein. In one embodiment, step B may also include adding a 4-1BB agonist to the medium as described elsewhere herein. In one embodiment, step B may also comprise adding an OX-40 agonist to the medium as described elsewhere herein. In addition, additives such as peroxisome proliferator activated receptor gamma coactivator I-alpha-agonists including a proliferation activated receptor (PPAR) -gamma agonist such as a thiazolidinedione compound may be used in the medium during step B as described in U.S. patent application publication No. US 2019/0307796 A1 (the disclosure of which is incorporated herein by reference in its entirety).

C. Step C: transition from initial first amplification to rapid second amplification

In certain instances, a population of bulk TILs obtained from an initial first amplification (which may include an amplification sometimes referred to as prep) including, for example, a population of TILs obtained from step B, e.g., as shown in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C), 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 discussed below. Similarly, in a situation in which a genetically modified TIL is to be used in therapy, the amplified population of TILs from the initial first amplification or the amplified population of TILs from the rapid second amplification may be genetically modified for suitable 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 an initial first amplification (e.g., step B shown in fig. 1 (and in particular, e.g., fig. 1B and/or fig. 8C)) is stored until phenotypes are determined for selection. In some embodiments, TIL obtained from an initial first amplification (e.g., step B shown in fig. 1 (and in particular, e.g., fig. 1B and/or fig. 8C)) is not stored and is directly subjected to a rapid second amplification. In some embodiments, TIL obtained from the initial first amplification is not cryopreserved after the initial first amplification and before the rapid second amplification. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after tumor disruption occurs and/or about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after the disruption occurs and/or about 3 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after the disruption occurs and/or about 3 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or about 4 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or about 4 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or about 5 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or about 5 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or about 6 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or about 6 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after the disruption occurs and/or about 7 days to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after the disruption occurs and/or about 7 days after the initial first amplification step is initiated. In some embodiments, the transition from the initial first TIL amplification to the second amplification occurs after the disruption occurs and/or about 8 days after the initial first amplification step is initiated.

In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after the disruption occurs and/or 1, 2, 3, 4, 5, 6, 7, or 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or 1 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or 1 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or 2 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or 2 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or 3 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to second amplification occurs after fragmentation occurs and/or 3 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or 4 to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or 4 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or from 5 days to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or from 5 days to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or from 6 days to 7 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or from 6 days to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after fragmentation occurs and/or 7 to 8 days after initiation of the initial first amplification step. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs 7 days after the disruption occurs and/or after the initial first amplification step is initiated. In some embodiments, the transition from initial first TIL amplification to rapid second amplification occurs after the disruption occurs and/or 8 days after initiation of the initial first amplification step.

In some embodiments, the TIL is not stored after the primary first amplification (primary first expansion) and prior to the rapid second amplification, and the TIL is directly subjected to the rapid second amplification (e.g., in some embodiments, is not stored during the transition from step B to step D as shown in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, TIL from the initial first amplification (second population of TILs) is directly subjected to the rapid second amplification without a transition phase.

In some embodiments, the transition from the initial first amplification to the rapid second amplification (e.g., according to step C of fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C)) 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 employed is, for example, GREX-10 or GREX-100. In some embodiments, the closed system bioreactor is a single bioreactor. In some embodiments, the transition from the initial 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 collection and initial first expansion (step a and step B) and transition called step C as shown in fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C). This further amplification is referred to herein as 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; such as the process shown in FIG. 8 (and in particular, e.g., step D of FIG. 8B)). Rapid second expansion is typically accomplished in a gas-permeable vessel using a medium that contains some 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., day 8, 9, 10, or 11 of the entire 3 rd generation process).

In some embodiments, the rapid second amplification of TIL (which may include amplification sometimes referred to as REP; and the process as shown in FIG. 1 (and specifically, e.g., FIG. 1B and/or FIG. 8C) step D) may be performed using any TIL flask or vessel known to those skilled 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 2 days after initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 3 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 4 days after initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 5 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 6 days after initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 7 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 8 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 9 days after the initiation of the rapid second amplification. In some embodiments, the second TIL amplification may be performed about 10 days after initiation of the rapid second amplification.

In one embodiment, the rapid 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 as shown in FIG. 8 (particularly, for example, the process shown in FIG. 8B and/or FIG. 8C) step D. 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 that of feeder cells present in the initial first expansion. For example, TIL may 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 stimulation may include, for example, anti-CD 3 antibodies, such as OKT3 of about 30ng/mL, mouse monoclonal anti-CD 3 antibodies (available from Ortho-McNeil (latin, new jersey) or Miltenyi Biotech (obu, california)), or UHCT-1 (available from BioLegend, san diego, california). TIL can be amplified by including more than one antigen of cancer (including antigenic portions thereof such as epitopes) during the second amplification to induce further in vitro stimulation of TIL, optionally expressed from a vector, e.g., human white blood cell 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 such as 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 HLA-A2 expressing antigen presenting cells. Alternatively, the TIL may be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of the second amplification. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In one embodiment, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL OKT-3 antibody. In one embodiment, 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 of the OKT-3 antibody. In one embodiment, the cell culture medium comprises 0.1ng/mL to 1ng/mL, 1ng/mL to 5ng/mL, 5ng/mL to 10ng/mL, 10ng/mL to 20ng/mL, 20ng/mL to 30ng/mL, 30ng/mL to 40ng/mL, 40ng/mL to 50ng/mL, and 50ng/mL to 100ng/mL OKT-3 antibodies. In one embodiment, the cell culture medium comprises between 15ng/mL and 30ng/mL OKT-3 antibody. In one embodiment, the cell culture medium comprises 30ng/mL to 60ng/mL OKT-3 antibody. In one embodiment, the cell culture medium comprises about 30ng/mL OKT-3. In one embodiment, the cell culture medium comprises about 60ng/mL OKT-3. In some embodiments, the OKT-3 antibody is Moromolizumab.

In some embodiments, the medium of rapid second amplification comprises IL-2. In some embodiments, the medium contains 6000IU/mL IL-2. In some embodiments, the rapid second expansion medium comprises antigen presenting feeder cells. In some embodiments, the medium for rapid second amplification comprises 7.5X10 per vessel ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium for rapid second amplification comprises OKT-3. In some embodiments, the medium for rapid second amplification comprises 500mL of medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100MCS flask. In some embodiments, the medium for rapid second amplification comprises 6000IU/mL IL-2, 60ng/mL OKT-3, and 7.5X10 ⁸ Individual antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium per container, and 6000IU/mL of IL-2, 30 μg of OKT-3, and 7.5X10 ⁸ Individual antigen presenting feeder cells.

In some embodiments, the medium of rapid second amplification comprises IL-2. In some embodiments, the medium contains 6000IU/mL IL-2. In some embodiments, the medium of the rapid second amplification comprises antigen presentation Feeder cells. In some embodiments, the medium comprises between 5X 10 per container ⁸ And 7.5X10 ⁸ Antigen presenting feeder cells between individuals. In some embodiments, the medium for rapid second amplification comprises OKT-3. In some embodiments, the medium for rapid second amplification comprises 500mL of medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100MCS flask. In some embodiments, the medium for rapid second amplification comprises 6000IU/mL IL-2, 60ng/mL OKT-3, and 5X 10 ⁸ To 7.5X10 ⁸ Individual antigen presenting feeder cells. In some embodiments, the rapid second amplification medium comprises 500mL of medium per container, and 6000IU/mL of IL-2, 30 μg of OKT-3, and 5X 10 ⁸ To 7.5X10 ⁸ Individual antigen presenting feeder cells.

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 step D process according to FIG. 8 (particularly, e.g., FIG. 8B and/or FIG. 8C) and as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is used 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 step D of FIG. 8 (specifically, e.g., FIG. 8B and/or FIG. 8C), and as described herein.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TIL to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1 to 10, about 1 to 15, about 1 to 20, about 1 to 25, about 1 to 30, about 1 to 35, about 1 to 40, about 1 to 45, about 1 to 50, about 1 to 75, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In one embodiment, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1 and 50 and 1 to 300. In one embodiment, the ratio of TIL to PBMCs in the rapid amplification and/or the second amplification is between 1 to 100 and 1 to 200.

In one embodiment, REP and/or flash secondary expansion is performed in culture flasks, wherein bulk TIL is mixed with 100 or 200 fold excess of deactivated feeder cells, 30ng/mL OKT3 anti-CD 3 antibody, and 6000IU/mL IL-2 in 150mL medium, wherein the feeder cell concentration is at least 1.1 fold (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 2X, 2.1X2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, or 4.0X of the feeder cell concentration in the initial primary expansion. Replacement medium (2/3 medium is typically replaced by pumping 2/3 of the used medium and replacing it with an equal volume of fresh medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable vessels 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 discussed in the examples and figures. 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 one embodiment, the second amplification (which may include amplification called REP, and FIG. 8 (particularly, e.g., those referred to in step D of FIG. 8B and/or FIG. 8C)) may be performed in 500mL capacity gas permeable flasks (G-Rex 100, available from Wilson Wolf Manufacturing Corporation, new Britton, minnesota, USA) with a 100cm gas permeable silicon bottom, 5X 10 ⁶ Or 10X 10 ⁶ TIL can be cultured with PBMC 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℃with 5% CO ₂ And (3) incubating. On day 5, 250mL of supernatant may be removed and placed in a centrifuge bottle and centrifuged at 1500rpm (491 Xg) for 10 minutes. The TIL pellet can 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 can be moved to a larger flask, such as GREX-500, on day 10 or 11. Cells may be collected 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 replacement medium is performed until the cells are transferred to an alternative growth chamber. In some embodiments, 2/3 of the medium is replaced by pumping spent medium and replacing it with an equal volume of fresh medium. In some embodiments, the alternative growth chamber includes a GREX flask and a gas permeable container as discussed more fully below.

In some embodiments, the serum-free or defined medium comprises basal cell culture medium, serum supplements and/or serum substitutes. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS ^TM OpTmizer ^TM T cell expansion basal medium, CTS ^TM OpTmizer ^TM T cell expansion SFM, CTS ^TM AIM-V Medium, CTS ^TM AIM-V SFM、LymphoONE ^TM T cell expansion Xeno-free medium, dulbecco's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (aMEM), glasgow minimal essential medium (G-MEM), RPMI growth medium, and Iscove's modified Dulbecco's medium.

In some embodiments, the serum supplement or serum replacement includes, but is not limited to, one or more CTS ^TM Optmizer T cell expansion 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 medium is determined to comprise 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 a pharmaceutical composition containing the sameHas trace element part Ag ⁺ 、Al ³⁺ 、Ba ²⁺ 、Cd ²⁺ 、Co ²⁺ 、Cr ³⁺ 、Ge ⁴⁺ 、Se ⁴⁺ 、Br、T、Mn ²⁺ 、P、Si ⁴⁺ 、V ⁵⁺ 、Mo ⁶⁺ 、Ni ²⁺ 、Rb ⁺ 、Sn ²⁺ And Zr (Zr) ⁴⁺ Is a compound of (a). In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS ^TM OpTmizer ^TM The T cell immune cell serum replacement is used with conventional growth media including, but not limited to CTS ^TM OpTmizer ^TM T cell expansion basal medium, CTS ^TM OpTmizer ^TM T cell expansion SFM, CTS ^TM AIM-V medium, CST ^TM AIM-V SFM、LymphoONE ^TM T cell expansion Xeno-free medium, dulbecco's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (aMEM), glasgow minimal essential medium (G-MEM), RPMI growth medium, and Iscove's modified Dulbecco's medium.

In some embodiments, the serum-free or defined medium is CTS ^TM OpTmizer ^TM T cell expansion SFM (ThermoFisher Scientific). Any CTS ^TM OpTmizer ^TM Formulations are useful in the present invention. CTS (clear to send) ^TM OpTmizer ^TM T cell expansion SFM 1L CTS ^TM OpTmizer ^TM T cell expansion basal medium and 26mL CTS ^TM OpTmizer ^TM T cell expansion supplements are mixed together prior to use. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol.

In some embodiments, the medium is determined to be CTS ^TM OpTmizer ^TM T cell expansion SFM (ThermoFisher Scientific). Any CTS ^TM OpTmizer ^TM Formulations are useful in the present invention. CTS (clear to send) ^TM OpTmizer ^TM T cell expansion SFM 1L CTS ^TM OpTmizer ^TM T cell expansion basal medium and 26mL CTS ^TM OpTmizer ^TM T cell expansion supplements are mixed together prior to use. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol and 2mM L-glutamine. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising from about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol, and 2mM L-glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immunocyte Serum Replacement (SR) (ThermoFisher Scientific), 55mM 2-mercaptoethanol and 2mM L-glutamine, further comprisesIL-2 at about 6000 IU/mL. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol, further comprising from about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol, further comprising about 3000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM 2-mercaptoethanol, further comprising IL-2 in an amount of about 1000IU/mL to about 6000 IU/mL. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, further comprising about 1000IU/mL to about 8000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, further comprising about 3000IU/mL IL-2. In some embodiments, CTS ^TM OpTmizer ^TM T cell expanded SFM was supplemented with about 3% CTS ^TM Immune cell Serum Replacement (SR) (ThermoFisher Scientific) 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 5 to about 150mM, 10 to about 140mM, 15 to about 130mM, 20 to about 120mM, 25 to about 110mM, 30 to about 100mM, 35 to about 95mM, 40 to about 90mM, 45 to about 85mM, 50 to about 80mM, 55 to about 75mM, 60 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 defined media described in International patent application publication No. WO 1998/030679 and U.S. patent application publication No. US 2002/0076747 A1 (which are incorporated herein by reference in their entirety) may be used in the present invention. In this publication, 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. Serum-free eukaryotic cell culture medium supplements comprise or are 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 medium is determined to comprise 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, and L-perishable Hemic acid-2-phosphate, iron-saturated transferrin, insulin and Ag containing trace element fraction ⁺ 、Al ³⁺ 、Ba ²⁺ 、Cd ²⁺ 、Co ²⁺ 、Cr ³⁺ 、Ge ⁴⁺ 、Se ⁴⁺ 、Br、T、Mn ²⁺ 、P、Si ⁴⁺ 、V ⁵ ⁺ 、Mo ⁶⁺ 、Ni ²⁺ 、Rb ⁺ 、Sn ²⁺ And Zr (Zr) ⁴⁺ Is a compound of (a). In some embodiments, the basal cell culture medium is selected from the group consisting of: dulbecco's Modified Eagle's Medium (DMEM), minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (. Alpha.MEM), glasgow minimal essential medium (G-MEM), RPMI growth medium, and Iscove's modified Dulbecco's medium.

In some embodiments, the glycine concentration in the medium is determined to be about 5 to 200mg/L, the L-histidine concentration is about 5 to 250mg/L, the L-isoleucine concentration is about 5 to 300mg/L, the L-methionine concentration is about 5 to 200mg/L, the L-phenylalanine concentration is about 5 to 400mg/L, the L-proline concentration is about 1 to 1000mg/L, the L-hydroxyproline concentration is about 1 to 45mg/L, the L-serine concentration is about 1 to 250mg/L, the L-threonine concentration is about 10 to 500mg/L, the L-tryptophan concentration is about 2 to 110mg/L, the L-tyrosine concentration is about 3 to 175mg/L, the L-valine concentration is about 5 to 500mg/L, the thiamine concentration is about 1 to 20mg/L, the reduced glutathione concentration is about 1 to 20mg/L, the L-ascorbic acid-2-phosphate concentration is about 1 to 200mg/L, the iron-saturated transferrin concentration is about 1 to 250mg/L, the L-transferrin concentration is about 1 to 50mg/L, the selenium concentration is about 0.0001 to 0.0001mg/L (e.g., about 0.0001mg/L, 0.00000 mg/L) I) The concentration is about 5000 to 50,000mg/L.

In some embodiments, the non-trace element fraction component of the defined medium is present in the concentration range listed in the column entitled "concentration range in 1X medium" in table 4. In other embodiments, the non-trace element fraction component of the defined medium is present at the final concentrations listed in the column entitled "preferred embodiment of medium 1X" in table 4. 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 the column entitled "preferred embodiments of supplement" in table 4.

In some embodiments, defined media described in Smith et al Clin Transl Immunology,2015,4 (1), e31 (the disclosure of which is incorporated herein by reference in its entirety) may be used in the present invention. Briefly, RPMI or CTS ^TM OpTmizer ^TM To be used as basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS ^TM Immune cell serum replacement.

In one embodiment, a rapid second amplification (including an amplification called REP) is performed and further comprises 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 2016/0010058 A1, the disclosure of which is incorporated herein by reference in its entirety, may be used to select TILs that are excellent in 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 assays can be performed on samples of bulk TIL that selectively mark dead cells and allow viability assessment. In some embodiments, TIL samples can be counted and assayed for viability using a Cellometer K2 automated cell counter (Nexcelom Bioscience, larens, ma). In some embodiments, viability is determined according to a standard cell counter K2Image Cytometer automated cell counter protocol.

In some embodiments, the rapid 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 rapid second amplification cultureThe medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises 6000IU/mL IL-2, 30 ug/flask OKT-3 and 7.5X10 as discussed in more detail below ⁸ Individual antigen presenting feeder cells (APCs). In some embodiments, the rapid 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 rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises 6000IU/mL IL-2, 30 ug/flask OKT-3, and 5X 10 as discussed in more detail below ⁸ Individual antigen presenting feeder cells (APCs).

In some embodiments, the rapid second amplification (e.g., according to step D) of fig. 8 (in particular, e.g., fig. 8B and/or fig. 8C) 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.

1. Feeder cells and antigen presenting cells

In one embodiment, the rapid second amplification procedure described herein (e.g., including those such as described in fig. 8 (and specifically, e.g., fig. 8B and/or 8C) step D 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 Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units obtained from healthy blood donors. PBMCs were 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 surviving cells on day 7 or 14 is less than the initial number of surviving 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 the TIL expansion procedure described herein if the total number of surviving cells on days 7 and 14 of culture in the presence of OKT3 and IL-2 is not increased compared to the initial number of surviving 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 the TIL expansion procedure described herein if the total number of surviving cells on days 7 and 14 of culture in the presence of OKT3 and IL-2 is not increased compared to the initial number of surviving 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, the PBMCs 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 one embodiment, the ratio of TIL to antigen presenting feeder cells in the second expansion is about 1 to 10, about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In one embodiment, the proportion of TIL to antigen presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In one embodiment, the proportion of TIL to antigen presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.

In one embodiment, the second amplification procedure described herein requires about 5×10 ⁸ Pairs of individual feeder cells were approximately 100X 10 ⁶ Proportion of individual TILs. In one embodiment, the second amplification procedure described herein requires about 7.5X10 ⁸ Pairs of individual feeder cells were approximately 100X 10 ⁶ Proportion of individual TILs. In another embodiment, the second amplification procedure described herein requires about 5X 10 ⁸ Pairs of individual feeder cells were approximately 50X 10 ⁶ Proportion of individual TILs. In another embodiment, the second amplification procedure described herein requires about 7.5X10 ⁸ Pairs of individual feeder cells were approximately 50X 10 ⁶ Proportion of individual TILs. In yet another embodiment, the second amplification procedure described herein requires about 5×10 ⁸ Pairs of individual feeder cells were approximately 25X 10 ⁶ And TIL. In yet another embodiment, the second amplification procedure described herein requires about 7.5X10 ⁸ Pairs of individual feeder cells were approximately 25X 10 ⁶ And TIL. In yet another embodiment, the rapid second expansion requires twice the number of feeder cells as the rapid second expansion. In yet another embodiment, about 2.5X10 are required for the initial first amplification as described herein ⁸ In the case of feeder cells, about 5X 10 is required for rapid second expansion ⁸ And (3) feeder cells. In yet another embodiment, about 2.5X10 are required for the initial first amplification as described herein ⁸ In the case of feeder cells, about 7.5X10 are required for rapid second expansion ⁸ And (3) feeder cells. In yet another embodiment, the rapid second expansion requires twice the number (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X of feeder cells as the initial first expansion.

In one embodiment, 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 Peripheral Blood Mononuclear Cells (PBMCs) of standard whole blood units obtained from allogeneic healthy blood donors. PBMCs were obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting (aAPC) cells are used in place of PBMCs. In some embodiments, PBMCs are added to the rapid second amplification at twice the concentration of PBMCs added to the initial first amplification.

In one embodiment, artificial antigen presenting cells are used in the rapid second expansion to replace or in combination with PBMCs.

2. Cytokines and other additives

The rapid second amplification method described herein typically uses a medium with a high dose of cytokines (specifically IL-2), as is known in the art.

Alternatively, it is additionally possible to use a combination of cytokines for rapid second amplification of TIL, such as a combination of two or more of IL-2, IL-15 and IL-21 as described in U.S. patent application publication No. US 2017/0107490 A1, the disclosure of which is incorporated herein by reference in its entirety. 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 lymphocyte production, particularly the T cells described therein.

In one embodiment, step D may also comprise adding OKT-3 antibody or moromiab to the medium as described elsewhere herein. In one embodiment, step D may also include adding a 4-1BB agonist to the medium as described elsewhere herein. In one embodiment, step D may also comprise adding an OX-40 agonist to the medium as described elsewhere herein. In addition, additives such as peroxisome proliferator activated receptor gamma coactivator I-alpha-agonists including a proliferation activated receptor (PPAR) -gamma agonist such as a thiazolidinedione compound may be used in the medium during step D, as described in U.S. patent application publication No. US 2019/0307796 A1 (the disclosure of which is incorporated herein by reference in its entirety).

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 than four amplification steps, such as provided in fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C). In some embodiments, TIL is collected after two amplification steps, such as provided in fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C). 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 (specifically, e.g., fig. 8B and/or fig. 8C).

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 employed in the present process. In some embodiments, the TIL is collected using an automated system.

In some embodiments, step E according to fig. 8 (particularly, e.g., fig. 8B and/or 8C) is performed according to the processes described herein. In some embodiments, the containment system is accessed through a syringe under sterile conditions to maintain the sterility and containment characteristics of the system. In some embodiments, the containment systems described herein are employed.

In some embodiments, TIL is collected according to the methods described herein. In some embodiments, TIL between days 14 and 16 is collected using the methods described herein. In some embodiments, TIL is collected at 14 days using the methods described herein. In some embodiments, TIL is collected over 15 days using the methods described herein. In some embodiments, TIL is collected over 16 days using the methods described herein.

F. Step F: final formulation and transfer to infusion container

After steps a through E are completed as provided in an exemplary sequence in fig. 8 (and in particular, e.g., fig. 8B) and as detailed above and herein, the cells are transferred to a container (e.g., an infusion bag or sterile vial) for administration to a patient. In some embodiments, once a therapeutically sufficient amount of TIL is obtained using the amplification methods described above, they are transferred to a container for administration to a patient.

In one embodiment, the TIL amplified using the methods of the present disclosure is administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TIL in a sterile buffer. The TIL amplified as disclosed herein may be administered by any suitable route 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.

V. further embodiments of the 2 nd, 3 rd and other TIL manufacturing processes

This section describes alternative embodiments of the 2 nd, 3 rd and other TIL manufacturing processes, which can be used with CCR, chemokine receptors and other embodiments of the invention.

PBMC feeder cell ratio

In some embodiments, the medium used in the amplification methods described herein (see, e.g., fig. 8 (particularly, e.g., fig. 8B and/or fig. 8C)) 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, J.Immunol.1985,135,1719, incorporated herein by reference in its entirety.

In one embodiment, the number of PBMC feeder cell layers is calculated as follows:

volume of A.T cells (diameter 10 μm): v= (4/3) pi r ³ ＝523.6μm ³

B. G-Rex 100 (M) column with a height of 40 μm (4 cells): v= (4/3) pi r ³ ＝4×10 ¹² μm ³

C. Cell number required to fill column B: 4X 10 ¹² μm ³ /523.6μm ³ ＝7.6×10 ⁸ μm ³ *0.64＝4.86×10 ⁸

D. Cell number that can be optimally activated in 4D space: 4.86×10 ⁸ /24＝20.25×10 ⁶

E. Number of feeder cells and TIL extrapolated to G-Rex 500: TIL: 100X 10 ⁶ And feeder cells: 2.5X10 ⁹

In this calculation, the method was used with a distance of 100cm ² The cylinder of the substrate provides the approximate number of monocytes required for the TIL-activated icosahedral geometry. Calculated to be about 5×10 ⁸ Experimental junction of (2)The result is a T cell activation threshold that closely reflects NCI experimental data, such as Jin et al, J.Immunother.2012,35,283-292. In (C), the multiplier (0.64) is the equivalent sphere random packing density as calculated by Jaeger and Nagel, science,1992,255,1523-3. In (D), divisor 24 is the number of equivalent spheres in 4-dimensional space that can contact similar objects or "newton's number" as described by Musin, russ.

In one embodiment, the number of exogenously supplied antigen presenting feeder cells during the initial 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 performing the initial first expansion in a cell culture medium comprising about 50% less antigen presenting cells as compared to a rapid second expansion cell culture medium.

In another embodiment, 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 initial first amplification.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to just or about 20: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to just or about 10: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 9: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to just or about 8: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to just or about 7: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 6: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 5: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to just or about 4: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to just or about 3: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.9: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.8: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.7: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.6: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.5: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.4: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.3: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.2: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.1: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to just or about 10: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 5: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to just or about 4: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to just or about 3: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.9: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.8: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.7: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.6: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.5: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.4: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.3: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.2: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 2:1 to exactly or about 2.1: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the initial first amplification is exactly or about 2:1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during the initial first amplification 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 another embodiment, the number of APCs exogenously supplied during the initial first amplification is exactly or about 1X 10 ⁸ 、1.1×10 ⁸ 、1.2×10 ⁸ 、1.3×10 ⁸ 、1.4×10 ⁸ 、1.5×10 ⁸ 、1.6×10 ⁸ 、1.7×10 ⁸ 、1.8×10 ⁸ 、1.9×10 ⁸ 、2×10 ⁸ 、2.1×10 ⁸ 、2.2×10 ⁸ 、2.3×10 ⁸ 、2.4×10 ⁸ 、2.5×10 ⁸ 、2.6×10 ⁸ 、2.7×10 ⁸ 、2.8×10 ⁸ 、2.9×10 ⁸ 、3×10 ⁸ 、3.1×10 ⁸ 、3.2×10 ⁸ 、3.3×10 ⁸ 、3.4×10 ⁸ Or 3.5X10 ⁸ The number of APCs exogenously supplied during the rapid second amplification was exactly or about 3.5X10 ⁸ 、3.6×10 ⁸ 、3.7×10 ⁸ 、3.8×10 ⁸ 、3.9×10 ⁸ 、4×10 ⁸ 、4.1×10 ⁸ 、4.2×10 ⁸ 、4.3×10 ⁸ 、4.4×10 ⁸ 、4.5×10 ⁸ 、4.6×10 ⁸ 、4.7×10 ⁸ 、4.8×10 ⁸ 、4.9×10 ⁸ 、5×10 ⁸ 、5.1×10 ⁸ 、5.2×10 ⁸ 、5.3×10 ⁸ 、5.4×10 ⁸ 、5.5×10 ⁸ 、5.6×10 ⁸ 、5.7×10 ⁸ 、5.8×10 ⁸ 、5.9×10 ⁸ 、6×10 ⁸ 、6.1×10 ⁸ 、6.2×10 ⁸ 、6.3×10 ⁸ 、6.4×10 ⁸ 、6.5×10 ⁸ 、6.6×10 ⁸ 、6.7×10 ⁸ 、6.8×10 ⁸ 、6.9×10 ⁸ 、7×10 ⁸ 、7.1×10 ⁸ 、7.2×10 ⁸ 、7.3×10 ⁸ 、7.4×10 ⁸ 、7.5×10 ⁸ 、7.6×10 ⁸ 、7.7×10 ⁸ 、7.8×10 ⁸ 、7.9×10 ⁸ 、8×10 ⁸ 、8.1×10 ⁸ 、8.2×10 ⁸ 、8.3×10 ⁸ 、8.4×10 ⁸ 、8.5×10 ⁸ 、8.6×10 ⁸ 、8.7×10 ⁸ 、8.8×10 ⁸ 、8.9×10 ⁸ 、9×10 ⁸ 、9.1×10 ⁸ 、9.2×10 ⁸ 、9.3×10 ⁸ 、9.4×10 ⁸ 、9.5×10 ⁸ 、9.6×10 ⁸ 、9.7×10 ⁸ 、9.8×10 ⁸ 、9.9×10 ⁸ Or 1X 10 ⁹ And (3) APC.

In another embodiment, the number of APCs exogenously supplied during the initial first amplification is selected from exactly or about 1.5X10 ⁸ To exactly or about 3X 10 APC ⁸ The number of APCs supplied exogenously during the rapid second amplification is selected from the range of just or about 4X 10 APCs ⁸ To exactly or about 7.5X10 APC ⁸ Ranges of APC.

In another embodiment, the number of APCs exogenously supplied during the initial first amplification is selected from just or about 2X 10 ⁸ To exactly or about 2.5X10 APC ⁸ The number of APCs supplied exogenously during the rapid second amplification is selected from the range of just or about 4.5X10 ⁸ To exactly or about 5.5X10 APC ⁸ Ranges of APC.

In another embodiment, the number of APCs exogenously supplied during the initial first amplification is exactly or about 2.5X10 ⁸ The number of APCs exogenously supplied during the rapid second amplification was exactly or about 5X 10 ⁸ And (3) APC.

In one embodiment, the number of APCs (including, e.g., PBMCs) added on day 0 of initial first amplification is about half the number of PBMCs added on day 7 of initial 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 initial 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 initial first expansion (e.g., day 7 of the method).

In another embodiment, 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 initial first amplification.

In another embodiment, the APCs supplied at the initial first amplification source are selected from just or about 1.0X10 ⁶ APC/cm ² To exactly or about 4.5 x 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied at the initial first amplification source are selected from just or about 1.5X10 ⁶ APC/cm ² To just or about 3.5X10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied at the initial first amplification source are selected from just or about 2X 10 ⁶ APC/cm ² To just or about 3X 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs are supplied at the initial first amplification source at exactly or about 2X 10 ⁶ APC/cm ² Is inoculated in culture flasks.

In another embodiment, the APCs are supplied at exactly or about 1.0X10 s at the initial first amplification source ⁶ 、1.1×10 ⁶ 、1.2×10 ⁶ 、1.3×10 ⁶ 、1.4×10 ⁶ 、1.5×10 ⁶ 、1.6×10 ⁶ 、1.7×10 ⁶ 、1.8×10 ⁶ 、1.9×10 ⁶ 、2×10 ⁶ 、2.1×10 ⁶ 、2.2×10 ⁶ 、2.3×10 ⁶ 、2.4×10 ⁶ 、2.5×10 ⁶ 、2.6×10 ⁶ 、2.7×10 ⁶ 、2.8×10 ⁶ 、2.9×10 ⁶ 、3×10 ⁶ 、3.1×10 ⁶ 、3.2×10 ⁶ 、3.3×10 ⁶ 、3.4×10 ⁶ 、3.5×10 ⁶ 、3.6×10 ⁶ 、3.7×10 ⁶ 、3.8×10 ⁶ 、3.9×10 ⁶ 、4×10 ⁶ 、4.1×10 ⁶ 、4.2×10 ⁶ 、4.3×10 ⁶ 、4.4×10 ⁶ Or 4.5X10 ⁶ APC/cm ² Is inoculated in culture flasks.

In another embodiment, the APCs supplied at the second rapid amplification source are selected from the group consisting of just or about 2.5X10 ⁶ APC/cm ² To just or about 7.5 x 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied at the rapid second amplification source are selected from the group consisting of just or about 3.5X10 ⁶ APC/cm ² To about 6.0X10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied at the second rapid amplification source are selected from the group consisting of just or about 4.0X10 ⁶ APC/cm ² To about 5.5X10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied at the second rapid amplification source are selected from the group consisting of just or about 4.0X10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs are supplied at the second rapid amplification source at exactly or about 2.5X10 ⁶ APC/cm ² 、2.6×10 ⁶ APC/cm ² 、2.7×10 ⁶ APC/cm ² 、2.8×10 ⁶ 、2.9×10 ⁶ 、3×10 ⁶ 、3.1×10 ⁶ 、3.2×10 ⁶ 、3.3×10 ⁶ 、3.4×10 ⁶ 、3.5×10 ⁶ 、3.6×10 ⁶ 、3.7×10 ⁶ 、3.8×10 ⁶ 、3.9×10 ⁶ 、4×10 ⁶ 、4.1×10 ⁶ 、4.2×10 ⁶ 、4.3×10 ⁶ 、4.4×10 ⁶ 、4.5×10 ⁶ 、4.6×10 ⁶ 、4.7×10 ⁶ 、4.8×10 ⁶ 、4.9×10 ⁶ 、5×10 ⁶ 、5.1×10 ⁶ 、5.2×10 ⁶ 、5.3×10 ⁶ 、5.4×10 ⁶ 、5.5×10 ⁶ 、5.6×10 ⁶ 、5.7×10 ⁶ 、5.8×10 ⁶ 、5.9×10 ⁶ 、6×10 ⁶ 、6.1×10 ⁶ 、6.2×10 ⁶ 、6.3×10 ⁶ 、6.4×10 ⁶ 、6.5×10 ⁶ 、6.6×10 ⁶ 、6.7×10 ⁶ 、6.8×10 ⁶ 、6.9×10 ⁶ 、7×10 ⁶ 、7.1×10 ⁶ 、7.2×10 ⁶ 、7.3×10 ⁶ 、7.4×10 ⁶ Or 7.5X10 ⁶ APC/cm ² Is inoculated in culture flasks.

In another embodiment, the APCs are supplied at exactly or about 1.0X10 s at the initial first amplification source ⁶ 、1.1×10 ⁶ 、1.2×10 ⁶ 、1.3×10 ⁶ 、1.4×10 ⁶ 、1.5×10 ⁶ 、1.6×10 ⁶ 、1.7×10 ⁶ 、1.8×10 ⁶ 、1.9×10 ⁶ 、2×10 ⁶ 、2.1×10 ⁶ 、2.2×10 ⁶ 、2.3×10 ⁶ 、2.4×10 ⁶ 、2.5×10 ⁶ 、2.6×10 ⁶ 、2.7×10 ⁶ 、2.8×10 ⁶ 、2.9×10 ⁶ 、3×10 ⁶ 、3.1×10 ⁶ 、3.2×10 ⁶ 、3.3×10 ⁶ 、3.4×10 ⁶ 、3.5×10 ⁶ 、3.6×10 ⁶ 、3.7×10 ⁶ 、3.8×10 ⁶ 、3.9×10 ⁶ 、4×10 ⁶ 、4.1×10 ⁶ 、4.2×10 ⁶ 、4.3×10 ⁶ 、4.4×10 ⁶ Or 4.5X10 ⁶ APC/cm ² Is inoculated in culture flasks at a density of exactly or about 2.5X10 for APCs supplied exogenously in rapid second amplification ⁶ APC/cm ² 、2.6×10 ⁶ APC/cm ² 、2.7×10 ⁶ APC/cm ² 、2.8×10 ⁶ 、2.9×10 ⁶ 、3×10 ⁶ 、3.1×10 ⁶ 、3.2×10 ⁶ 、3.3×10 ⁶ 、3.4×10 ⁶ 、3.5×10 ⁶ 、3.6×10 ⁶ 、3.7×10 ⁶ 、3.8×10 ⁶ 、3.9×10 ⁶ 、4×10 ⁶ 、4.1×10 ⁶ 、4.2×10 ⁶ 、4.3×10 ⁶ 、4.4×10 ⁶ 、4.5×10 ⁶ 、4.6×10 ⁶ 、4.7×10 ⁶ 、4.8×10 ⁶ 、4.9×10 ⁶ 、5×10 ⁶ 、5.1×10 ⁶ 、5.2×10 ⁶ 、5.3×10 ⁶ 、5.4×10 ⁶ 、5.5×10 ⁶ 、5.6×10 ⁶ 、5.7×10 ⁶ 、5.8×10 ⁶ 、5.9×10 ⁶ 、6×10 ⁶ 、6.1×10 ⁶ 、6.2×10 ⁶ 、6.3×10 ⁶ 、6.4×10 ⁶ 、6.5×10 ⁶ 、6.6×10 ⁶ 、6.7×10 ⁶ 、6.8×10 ⁶ 、6.9×10 ⁶ 、7×10 ⁶ 、7.1×10 ⁶ 、7.2×10 ⁶ 、7.3×10 ⁶ 、7.4×10 ⁶ Or 7.5X10 ⁶ APC/cm ² Is inoculated in culture flasks.

In another embodiment, the APCs supplied exogenously in the initial first amplification are selected from exactly or about 1.0X10 ⁶ APC/cm ² To exactly or about 4.5 x 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density in the range of (1) and exogenously supplied APCs in the rapid second amplification are selected from the group consisting of just or about 2.5X10 ⁶ APC/cm ² To just or about 7.5 x 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied exogenously in the initial first amplification are selected from exactly or about 1.5X10 ⁶ APC/cm ² To just or about 3.5X10 ⁶ APC/cm ² Is inoculated in culture flasks at a density in the range of (3) 5X 10, selected from the group consisting of just or about ⁶ APC/cm ² To exactly or about 6X 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied exogenously in the initial first amplification are selected from exactly or about 2X 10 ⁶ APC/cm ² To just or about 3X 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density in the range of (3) and exogenously supplied APCs in the rapid second amplification are selected from the group consisting of just or about 4X 10 ⁶ APC/cm ² To exactly or about 5.5X10 ⁶ APC/cm ² Is inoculated in culture flasks at a density within the range of (2).

In another embodiment, the APCs supplied exogenously in the initial first amplification are at or about 2X 10 ⁶ APC/cm ² Is inoculated in culture flasks at a density of exactly or about 4X 10 for exogenously supplied APCs in the rapid second amplification ⁶ APC/cm ² Is inoculated in culture flasks.

In another embodiment, the ratio of the number of exogenously supplied APCs (including, e.g., PBMCs) on day 7 of rapid second amplification to the number of exogenously supplied PBMCs on day 0 of initial first amplification is selected from exactly or about 1.1:1 to just or about 20: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs (including, e.g., PBMCs) on day 7 of rapid second amplification to the number of exogenously supplied PBMCs on day 0 of initial first amplification is selected from exactly or about 1.1:1 to just or about 10: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs (including, e.g., PBMCs) on day 7 of rapid second amplification to the number of exogenously supplied PBMCs on day 0 of initial first amplification is selected from exactly or about 1.1:1 to exactly or about 9: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to just or about 8: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to just or about 7: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 6: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 5: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to just or about 4: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to just or about 3: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.9: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.8: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.7: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.6: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.5: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.4: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.3: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.2: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2.1: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of the initial first amplification is selected from exactly or about 1.1:1 to exactly or about 2: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to just or about 10: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 5: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to just or about 4: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to just or about 3: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.9: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.8: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.7: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.6: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.5: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.4: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.3: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.2: 1.

In another embodiment, 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 initial first amplification is selected from exactly or about 2:1 to exactly or about 2.1: 1.

In another embodiment, 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 initial first amplification is exactly or about 2:1.

in another embodiment, 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 initial first amplification 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 another embodiment, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initial first amplification is exactly or about 1X 10 ⁸ 、1.1×10 ⁸ 、1.2×10 ⁸ 、1.3×10 ⁸ 、1.4×10 ⁸ 、1.5×10 ⁸ 、1.6×10 ⁸ 、1.7×10 ⁸ 、1.8×10 ⁸ 、1.9×10 ⁸ 、2×10 ⁸ 、2.1×10 ⁸ 、2.2×10 ⁸ 、2.3×10 ⁸ 、2.4×10 ⁸ 、2.5×10 ⁸ 、2.6×10 ⁸ 、2.7×10 ⁸ 、2.8×10 ⁸ 、2.9×10 ⁸ 、3×10 ⁸ 、3.1×10 ⁸ 、3.2×10 ⁸ 、3.3×10 ⁸ 、3.4×10 ⁸ Or 3.5X10 ⁸ The number of APCs (including, e.g., PBMC) exogenously supplied on day 7 of rapid second amplification (including, e.g., PBMC) is exactly or about 3.5X10 ⁸ 、3.6×10 ⁸ 、3.7×10 ⁸ 、3.8×10 ⁸ 、3.9×10 ⁸ 、4×10 ⁸ 、4.1×10 ⁸ 、4.2×10 ⁸ 、4.3×10 ⁸ 、4.4×10 ⁸ 、4.5×10 ⁸ 、4.6×10 ⁸ 、4.7×10 ⁸ 、4.8×10 ⁸ 、4.9×10 ⁸ 、5×10 ⁸ 、5.1×10 ⁸ 、5.2×10 ⁸ 、5.3×10 ⁸ 、5.4×10 ⁸ 、5.5×10 ⁸ 、5.6×10 ⁸ 、5.7×10 ⁸ 、5.8×10 ⁸ 、5.9×10 ⁸ 、6×10 ⁸ 、6.1×10 ⁸ 、6.2×10 ⁸ 、6.3×10 ⁸ 、6.4×10 ⁸ 、6.5×10 ⁸ 、6.6×10 ⁸ 、6.7×10 ⁸ 、6.8×10 ⁸ 、6.9×10 ⁸ 、7×10 ⁸ 、7.1×10 ⁸ 、7.2×10 ⁸ 、7.3×10 ⁸ 、7.4×10 ⁸ 、7.5×10 ⁸ 、7.6×10 ⁸ 、7.7×10 ⁸ 、7.8×10 ⁸ 、7.9×10 ⁸ 、8×10 ⁸ 、8.1×10 ⁸ 、8.2×10 ⁸ 、8.3×10 ⁸ 、8.4×10 ⁸ 、8.5×10 ⁸ 、8.6×10 ⁸ 、8.7×10 ⁸ 、8.8×10 ⁸ 、8.9×10 ⁸ 、9×10 ⁸ 、9.1×10 ⁸ 、9.2×10 ⁸ 、9.3×10 ⁸ 、9.4×10 ⁸ 、9.5×10 ⁸ 、9.6×10 ⁸ 、9.7×10 ⁸ 、9.8×10 ⁸ 、9.9×10 ⁸ Or 1X 10 ⁹ Individual APCs (including, for example, PBMCs).

In another embodiment, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initial first amplification is selected from just or about 1X 10 ⁸ The number of APCs (including, for example, PBMC) is up to or about 3.5X10 ⁸ The range of APCs (including, e.g., PBMC) and the number of APCs (including, e.g., PBMC) exogenously supplied on day 7 of rapid second amplification is selected from just or about 3.5X10 ⁸ The number of APCs (including, for example, PBMC) is up to or about 1X 10 ⁹ Ranges of APCs (including, for example, PBMCs).

In another embodiment, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initial first amplification is selected from exactly or about 1.5X10 ⁸ To exactly or about 3X 10 APC ⁸ The number of APCs (including, e.g., PBMC) exogenously supplied on day 7 of rapid second amplification is selected from the group consisting of just or about 4X 10 APCs (including, e.g., PBMC) ⁸ The number of APCs (including, for example, PBMC) is up to or about 7.5X10 ⁸ Ranges of APCs (including, for example, PBMCs).

In another embodiment, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initial first amplification is selected from just or about 2X 10 ⁸ The number of APCs (including, for example, PBMC) is up to or about 2.5X10 ⁸ The range of APCs (including, e.g., PBMC) and the number of APCs (including, e.g., PBMC) exogenously supplied on day 7 of rapid second amplification is selected from just or about 4.5X10 ⁸ The number of APCs (including, for example, PBMC) is up to or about 5.5X10 ⁸ Ranges of APCs (including, for example, PBMCs).

In another embodiment, the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initial first amplification is exactly or about 2.5X10 ⁸ Number of APCs (including, e.g., PBMC) exogenously supplied on day 7 of rapid second amplificationIs exactly or about 5X 10 ⁸ Individual APCs (including, for example, PBMCs).

In one embodiment, the number of APC (including, e.g., PBMCs) added on day 0 of the initial 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 initial first expansion and adding an antigen presenting cell layer to the second TIL population on day 7, wherein the amount of antigen presenting cell layer added on day 0 is about 50% of the amount of antigen presenting cell layer added on day 7.

In another embodiment, 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 initial first amplification.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 4 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 3 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from about 1.5 cell layers to about 2.5 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of about 3 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about one cell layer, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 2 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMC) having an average thickness of exactly 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, day 7 of the rapid second amplification occurs in the presence of a layered (including, for example) layer of APC having an average thickness of exactly 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, 7.8, 7.9 or 8 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from about 1 cell layer to about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from about 3 cell layers to about 10 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from about 2 cell layers to about 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of from about 4 cell layers to about 8 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 2 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 4 cell layers to exactly or about 8 cell layers.

In another embodiment, day 0 of the initial first amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 1, 2, or 3 cell layers, and day 7 of the rapid second amplification occurs in the presence of layered APCs (including, e.g., PBMCs) having an average thickness of exactly or about 3, 4, 5, 6, 7, 8, 9, or 10 cell layers.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 10.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 8.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 7.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 6.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 5.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 4.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 3.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.1 to just or about 1: 2.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.2 to just or about 1: 8.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.3 to just or about 1: 7.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.4 to just or about 1: 6.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.5 to just or about 1: 5.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.6 to just or about 1: 4.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.7 to just or about 1: 3.5.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.8 to just or about 1:3.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 the group consisting of exactly or about 1:1.9 to just or about 1: 2.5.

In another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 exactly or about 1:2.

in another embodiment, day 0 of the initial first amplification occurs 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 the rapid second amplification occurs 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 exactly 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 about 1.0X10 ⁶ APC/cm ² To about 4.5X10 ⁶ APC/cm ² The number of APCs in the rapid second amplification is selected from the range of about 2.5X10 ⁶ APC/cm ² To about 7.5X10 ⁶ APC/cm ² Is not limited in terms of the range of (a).

In some embodiments, the number of APCs in the initial first amplification is selected from about 1.5X10 ⁶ APC/cm ² To about 3.5X10 ⁶ APC/cm ² The number of APCs in the rapid second amplification is selected from the range of about 3.5X10 ⁶ APC/cm ² To about 6.0X10 ⁶ APC/cm ² Is not limited in terms of the range of (a).

In some embodiments, the number of APCs in the initial first amplification is selected from about 2.0X10 ⁶ APC/cm ² To about 3.0X10 ⁶ APC/cm ² The number of APCs in the rapid second amplification is selected from the range of about 4.0X10) ⁶ APC/cm ² To about 5.5X10 ⁶ APC/cm ² Is not limited in terms of the range of (a).

B. Optional cell culture Medium Components

1. anti-CD 3 antibodies

In some embodiments, the medium used in the amplification methods described herein, including those referred to as REP, see, e.g., fig. 1 and 8 (and specifically, e.g., fig. 8B), 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, J.Immunol.1985,135,1719, incorporated herein by reference in its entirety.

Those skilled in the art will appreciate that 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 is used as Moromolizumab (available from Ortho-McNeil (Laritan, N.J.) or Miltenyi Biotech (Orthon, calif.). In some embodiments, the anti-CD 3 antibody (e.g., OKT-3) is added immediately after the tumor fragments or digests are added to the medium of the first air-permeable flask, bag, or other container during the prep phase or initial REP phase (of the 3 rd generation method).

Those skilled in the art will appreciate that 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 is used as Moromolizumab (available from Ortho-McNeil (Laritan, N.J.) or Miltenyi Biotech (Orthon, calif.).

2.4-1BB (CD 137) agonists

In one embodiment, the first expanded and/or the rapidly second expanded cell culture medium comprises a TNFRSF agonist. In one embodiment, 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), type (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subtype 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), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized or chimeric antibodies and antibody fragments, such as Fab fragments, F (ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the foregoing, and engineered versions of antibodies, such as scFv molecules that bind to 4-1 BB. In one embodiment, the 4-1BB agonist is an antigen binding protein of a fully human antibody. In one embodiment, 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 of the present disclosure 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 mucins (adnectins), 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, PLOS One 2013,8, e69677. In a preferred embodiment, 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 one embodiment, the 4-1BB agonist is EU-101 (eutillex co.ltd.), wutuzumab or wuruituzumab, or a fragment, derivative, conjugate, variant or biological analog thereof. In a preferred embodiment, the 4-1BB agonist is WUTumumab or WURuilumumab or a fragment, derivative, conjugate, variant or biological analog thereof.

In a preferred embodiment, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In a preferred embodiment, a multimeric 4-1BB agonist such as a trimeric or hexameric 4-1BB agonist (having three or six ligand binding domains) induces superior receptor (4-1 BBL) aggregation and formation of an internal cellular signaling complex compared to an agonistic monoclonal antibody that typically possesses two ligand binding domains. Trimers (trivalent) or hexamers (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, mol.cancer Therapeutics 2013,12,2735-47.

The agonistic 4-1BB antibodies and fusion proteins are known to induce a strong immune response. In a preferred embodiment, 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 eliminates 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, 4-1BB agonists are characterized as binding to human 4-1BB (SEQ ID NO: 40) with high affinity and agonistic activity. In one embodiment, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 40). In one embodiment, 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 5.

Table 5: amino acid sequence of 4-1BB antigen

In some embodiments, the compositions, processes, and methods comprise a K of about 100pM or less _D Binding to human or murine 4-1BB at a K of about 90pM or less _D Binding to human or murine 4-1BB at a K of about 80pM or less _D Binding to human or murine 4-1BB at a K of about 70pM or less _D Binding to human or murine 4-1BB at a K of about 60pM or less _D Binding to human or murine 4-1BB at a K of about 50pM or less _D Binding to human or murine 4-1BB at a K of about 40pM or less _D Binding to human or murine 4-1BB or K at about 30pM or less _D 4-1BB agonists that bind to human or murine 4-1 BB.

In some embodiments, the compositions, processes, and methods comprise at about 7.5×10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine 4-1BB at about 7.5X10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine 4-1BB at about 8X 10 ⁵ K of l/M.s or faster _{Association with} Binding to human or murine 4-1BB at about 8.5X10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine 4-1BB at about 9X 10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine 4-1BB at about 9.5X10 ⁵ K of 1/M.s or more _{Association with} Combined with human or murine 4-1BB or at about 1X 10 ⁶ K of 1/M.s or more _{Association with} 4-1BB agonists that bind to human or murine 4-1 BB.

In some embodiments, the compositionThe process and method include a process of about 2 x 10 ^-5 K of 1/s or less _{Dissociation of} Combined with human or murine 4-1BB at about 2.1X10 ^-5 K of 1/s or less _{Dissociation of} Combined with human or murine 4-1BB at about 2.2X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine 4-1BB at about 2.3X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine 4-1BB at about 2.4X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine 4-1BB at about 2.5X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine 4-1BB at about 2.6X10 ^-5 K of 1/s or less _{Dissociation of} Combined with human or murine 4-1BB or at about 2.7X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine 4-1BB at about 2.8X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine 4-1BB at about 2.9X10 ^-5 K of 1/s or less _{Dissociation of} Combined with human or murine 4-1BB or at about 3X 10 ^-5 K of 1/s or less _{Dissociation of} 4-1BB agonists that bind to human or murine 4-1 BB.

In some embodiments, the compositions, processes, and methods comprise an IC of about 10nM or less ₅₀ IC binding to human or murine 4-1BB at about 9nM or less ₅₀ IC binding to human or murine 4-1BB at about 8nM or less ₅₀ IC binding to human or murine 4-1BB at about 7nM or less ₅₀ IC binding to human or murine 4-1BB at about 6nM or less ₅₀ IC binding to human or murine 4-1BB at about 5nM or less ₅₀ IC binding to human or murine 4-1BB at about 4nM or less ₅₀ IC binding to human or murine 4-1BB at about 3nM or less ₅₀ IC binding to human or murine 4-1BB at about 2nM or less ₅₀ IC that binds to human or murine 4-1BB or is about 1nM or less ₅₀ 4-1BB agonists that bind to human or murine 4-1 BB.

In a preferred embodiment, the 4-1BB agonist is Ulmatiumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant or biological analog thereof. Wu Tumu mab is available from Pfizer, inc. Wu Tumu monoclonal antibody is immunoglobulin G2-lambda antibody [ Chile TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9,4-1B ]B, T cell antigen ILA, CD 137) ]Homo sapiens (fully human) monoclonal antibodies. The amino acid sequence of Wu Tumu mab is shown in table 6. Wu Tumu monoclonal antibodies comprise: glycosylation sites at Asn59 and Asn 292; in positions 22-96 (V) _H -V _L )、143-199(C _H 1-C _L )、256-316(C _H 2) And 362-420 (C) _H 3) Disulfide bonds within the heavy chain of (a); in positions 22'-87' (V) _H -V _L ) And 136'-195' (C) _H 1-C _L ) Disulfide bonds within the light chain of (a); 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 WO 2012/0325433 A1, the respective disclosures of which are incorporated herein by reference in their entirety. The preclinical characterization of Wu Tumu mab is described in Fisher et al, cancer immunology.&Immunother.2012,61,1721-33. Current clinical trials of ulipristal antibodies in a variety of blood and solid tumor indications include the national institutes of health (U.S. national Institutes of Health) clinicaltrias gov accession numbers NCT02444793, NCT01307267, NCT02315066 and NCT02554812.

In one embodiment, the 4-1BB agonist comprises a sequence consisting of SEQ ID NO:42 and the heavy chain given by SEQ ID NO: 43. In one embodiment, 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 one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 99% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 98% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 97% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 96% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:42 and SEQ ID NO:43, and a heavy chain and a light chain having at least 95% identity.

In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VR) of Wu Tumu mab. In one embodiment, the 4-1BB agonist heavy chain variable region (V _H ) Comprising SEQ ID NO:44, the 4-1BB agonist light chain variable region (V _L ) Comprising SEQ ID NO:45 and conservative amino acid substitutions thereof. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:44 and SEQ ID NO:45 has at least 99% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:44 and SEQ ID NO:45 has a V with at least 98% identity to the sequence shown in 45 _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:44 and SEQ ID NO:45 has at least 97% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:44 and SEQ ID NO:45 has a V with at least 96% identity to the sequence shown in 45 _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:44 and SEQ ID NO:45 has at least 95% identity of V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises an scFv antibody, which comprises a sequence corresponding to SEQ ID NO:44 and SEQ ID NO:45 has at least 99% identity V to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the 4-1BB agonist comprises a peptide having the sequence set forth in SEQ ID NO: 46. SEQ ID NO:47 and SEQ ID NO:48 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 49. SEQ ID NO:50 and SEQ ID NO:51, and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by the drug administration with reference to Wu Tumu monoclonal antibody. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody, the 4-1BB 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 biologic product that comprises one or more post-translational modifications as compared to the reference drug or reference biologic product that is wu-mumab. 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, which is the Ubbelohde mab. The 4-1BB agonist antibodies may be licensed by a pharmaceutical regulatory agency such as EMA in the United states FDA and/or European Union. In some embodiments, the biosimilar is a composition provided as 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, which is wuyimumab. In some embodiments, the biosimilar is a composition provided as 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, which is wuyimumab.

Table 6: amino acid sequence of a 4-1BB agonist antibody related to Wu Tumu mab

In a preferred embodiment, 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 monoclonal antibodies are available from Bristol-Myers Squibb, inc. Wu Ruilu the monoclonal antibody is immunoglobulin G4-kappa antibody [ Chile TNFRSF9 (tumor necrosis factor receptor superfamily Member 9,4-1BB, T cell antigen ILA, CD 137)]Homo sapiens (fully human) monoclonal antibodies. The amino acid sequence of Wu Ruilu mab is shown in table 7. Wu Ruilu monoclonal antibodies comprise: an N-glycosylation site at position 298 (and 298'); in positions 22-95 (V) _H -V _L )、148-204(C _H 1-C _L )、262-322(C _H 2) And 368-426 (C) _H 3) (and the intra-heavy chain disulfide bonds at positions 22"-95", 148"-204", 262"-322" and 368 "-426"); in positions 23'-88' (V) _H -V _L ) And 136'-196' (C) _H 1-C _L ) (and light chain intra-chain disulfide bonds 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 is described in U.S. Pat. nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference in their entirety. The preclinical and clinical features of Wu Ruilu mab are described in Segal et al, clin.cancer Res.2016, available from 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 one embodiment, the 4-1BB agonist comprises a sequence consisting of SEQ ID NO:52 and the heavy chain given by SEQ ID NO: 53. In one embodiment, 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 one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 99% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 98% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 97% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 96% identity. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:52 and SEQ ID NO:53 have heavy and light chains with at least 95% identity.

In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VR) of Wu Ruilu mab. In one embodiment, the 4-1BB agonist heavy chain variable region (V _H ) Comprising SEQ ID NO:54, the 4-1BB agonist light chain variable region (V _L ) Comprising SEQ ID NO:55 and conservative amino acid substitutions thereof. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:54 and SEQ ID NO:55 has at least 99% identity to V of the sequence shown in seq id no _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:54 and SEQ ID NO:55 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:54 and SEQ ID NO:55 has at least 97% identity to a sequence of V _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:54 and SEQ ID NO:55 has a V with at least 96% identity to the sequence shown in 55 _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises a sequence corresponding to the sequence of SEQ ID NO:54 and SEQ ID NO:55 has at least 95% identity to V of the sequence shown in seq id no _H And V _L A zone. In one embodiment, the 4-1BB agonist comprises an scFv antibody, said scFv antibody comprising a sequence which is identical to the sequence of SEQ ID NO:54 and SEQ ID NO:55 has at least 99% identity to V of the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the 4-1BB agonist comprises a peptide having the sequence set forth in SEQ ID NO: 56. SEQ ID NO:57 and SEQ ID NO:58 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 59. SEQ ID NO:60 and SEQ ID NO:61, and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by the drug administration with reference to Wu Ruilu monoclonal antibody. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody, the 4-1BB 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 biologic product that comprises one or more post-translational modifications as compared to the reference drug or reference biologic product, the reference drug or reference biologic product being 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, which is nivolumab. The 4-1BB agonist antibodies may be licensed by a pharmaceutical regulatory agency such as EMA in the United states FDA and/or European Union. In some embodiments, the biosimilar is a composition provided as 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, which is nivolumab. In some embodiments, the biosimilar is a composition provided as 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, which is nivolumab.

Table 7: amino acid sequence of a 4-1BB agonist antibody related to Wu Ruilu mab

In one embodiment, the 4-1BB agonist is selected from the group consisting of: 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621 PABX), 145501 (Leinco Technologies B591), antibodies produced by the cell line of accession number 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. US 2005/0095244 (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. 7,214,493, 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. 554271), antibodies disclosed in U.S. Pat. No. 4E9 or BMS. 4635 A.S. Pat. No. 4, antibodies disclosed in U.S. No. 4E9 or BMS. 4635, antibodies disclosed in U.S. Pat. No. 5 A. 4E9 or 4B-4635, antibodies disclosed in U.S. Pat. No. 4 A. 4 A.3 or 4E 3 or 4E-4638 (e.S. 4 A.S. respective to each of the respective BMS. No. 4638 and 45 A.S. 45 and 45); 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 WO 2012/177788, WO 2015/119923, and WO 2010/042433, and fragments, derivatives, conjugates, variants, or biological analogs thereof, wherein the disclosures of the foregoing patents or patent application publications are each incorporated herein by reference in their entirety.

In one embodiment, the 4-1BB agonist is international patent application publication nos. WO 2008/025516 A1, WO 2009/0071120 A1, WO 2010/003766 A1, WO 2010/010051 A1, and WO 2010/078966 A1; U.S. patent application publication Nos. US 2011/0027218 A1, US 2015/0126209 A1, US 2011/011494 A1, US 2015/0110834 A1 and US 2015/012610 A1; and 4-1BB agonistic fusion proteins 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 in their entirety.

In one embodiment, the 4-1BB agonist is a 4-1BB agonistic fusion protein, or a fragment, derivative, conjugate, variant, or biological analog thereof, as depicted by structure I-A (C-terminal Fc-antibody fragment fusion protein) or structure I-B (N-terminal Fc-antibody fragment fusion protein) (see FIG. 18). In structures I-A and I-B, the cylinders refer to the respective polypeptide binding domains. Structures 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 tumor necrosis factor superfamily member 9 (TNFSF 9)) or antibodies that bind 4-1BB, which TNFRSF binding domains fold to form a trivalent protein, which is then passed through IgG1-Fc (including CH3 and C _H 2 domain) and then serves to link together two trivalent proteins via disulfide bonds (small oblong), 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 linked by a linker _H And V _L A chain, which may comprise hydrophilic residues and Gly and Ser sequences providing softness, glu and Lys providing solubility. Any scFv domain design may be used, for example as described in de Marco, microbial Cell Factories,2011,10,44; ahmad et al, clin.&Dev.immunol.2012,980250; monnier et al, antibodies,2013,2,193-208; or those incorporated by reference elsewhere herein. The structure of this form of fusion protein is 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 in their entirety.

The amino acid sequences of the other polypeptide domains of structure I-A are given in FIG. 18, see Table 8. The Fc domain preferably comprises the complete constant domain (amino acids 17 to 230 of SEQ ID NO: 62), the complete hinge domain (amino acids 1 to 16 of SEQ ID NO: 62) or a part of the hinge domain (e.g.amino acids 4 to 16 of SEQ ID NO: 62). Preferred linkers for linking the C-terminal Fc antibody may be selected from the group consisting of SEQ ID NOs: 63 to SEQ ID NO:72 includes linkers suitable for fusing additional polypeptides.

Table 8: the amino acid sequence of the TNFRSF agonist fusion protein, including the 4-1BB agonist fusion protein, has the C-terminal Fc antibody fragment fusion protein design (Structure I-A)

The amino acid sequences of the other polypeptide domains of structure I-B are given in FIG. 18, see Table 9. If an Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein as in structure I-B, the sequence of the Fc module is preferably shown in SEQ ID NO:73, the linker sequence is preferably selected from the group consisting of SEQ ID NO:74 to SEQ ID NO: 76.

Table 9: the amino acid sequence of the TNFRSF agonist fusion protein, including the 4-1BB agonist fusion protein, has the design of the N-terminal Fc antibody fragment fusion protein (structure I-B)

In one embodiment, the 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, wu Ruilu, wu Tumu, a variable heavy and a variable light chain selected from the variable heavy and variable light chains set forth in table 10, any combination of the foregoing, and fragments, derivatives, conjugates, variants, and biological analogs thereof.

In one embodiment, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain comprising a 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain comprising a sequence according to SEQ ID NO: 77. In one embodiment, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain comprising a soluble 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain comprising a sequence according to SEQ ID NO: 78.

In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain being a polypeptide comprising a sequence that is complementary to each of SEQ ID NOs: 43 and SEQ ID NO:44 has a V with at least 95% identity to the sequence shown in 44 _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker. In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain being a polypeptide comprising a sequence that is complementary to each of SEQ ID NOs: 54 and SEQ ID NO:55 has at least 95% identity to V of the sequence shown in seq id no _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker. In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain being a fusion protein comprising a sequence of V each as set forth in Table 10 _H And V _L V having at least 95% identity to the sequence _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker.

Table 10: additional polypeptide domains useful as 4-1BB binding domains in fusion proteins or as scFv 4-1BB agonist antibodies

In one embodiment, the 4-1BB agonist is a 4-1BB agonistic 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, and further comprises an additional domain at the N-terminus and/or C-terminus, which additional domain is a Fab or Fc fragment domain. In one embodiment, the 4-1BB agonist is a 4-1BB agonistic 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-and/or C-terminus, which additional domain is a Fab or Fc fragment domain, each of which lacks a stalk region (which contributes to trimerization and provides some 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 one embodiment, the 4-1BB agonist is a 4-1BB agonistic single chain fusion polypeptide, which comprises (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, each of the soluble TNF superfamily cytokine domains lacking a stem region, the first and second peptide linkers independently having 3 to 8 amino acids in length, each TNF superfamily cytokine domain being a 4-1BB binding domain.

In one embodiment, the 4-1BB agonist is a 4-1BB agonistic scFv antibody, which comprises any of the foregoing V _H The domain is linked to any of the foregoing V _L A domain.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist antibody of BPS Bioscience (product number 79097-2, available from BPS Bioscience, san Diego, calif., U.S.A.). In one embodiment, the 4-1BB agonist is a 4-1BB agonist antibody of Creative Biolabs (product number MOM-18179, available from Creative Biolabs, shirley, N.Y..

OX40 (CD 134) agonist

In one embodiment, 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), type (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) or subtype 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, humanized or chimeric antibodies and antibody fragments, such as Fab fragments, F (ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the foregoing, and engineered versions of antibodies, such as scFv molecules that bind to OX 40. In one embodiment, the OX40 agonist is an antigen binding protein of a fully human antibody. In one embodiment, 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 mucins, 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 a preferred embodiment, 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 a preferred embodiment, 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, j.immunoother.2009, 182,1481-89. In a preferred embodiment, a multimeric OX40 agonist such as a trimeric or hexameric OX40 agonist (having three or six ligand binding domains) induces superior receptor (OX 40L) aggregation and internal cellular signaling complex formation compared to an agonistic monoclonal antibody that typically possesses two ligand binding domains. Trimers (trivalent) or hexamers (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, mol.cancer Therapeutics 2013,12,2735-47.

Agonistic OX40 antibodies and fusion proteins are known to induce strong immune responses. Curti et al, cancer Res.2013,73,7189-98. In a preferred embodiment, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to an OX40 antigen in a manner sufficient to reduce 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 eliminates Fc region functionality.

In some embodiments, the OX40 agonist is characterized as binding to human OX40 (SEQ ID NO: 85) with high affinity and agonistic activity. In one embodiment, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 85). In one embodiment, the OX40 agonist is a binding molecule that binds to murine OX40 (SEQ ID NO: 86). The amino acid sequences of OX40 agonists or OX40 antigens to which the binding molecules bind are summarized in table 11.

Table 11: amino acid sequence of OX40 antigen

In some embodiments, the compositions, processes, and methods comprise a K of about 100pM or less _D Binds to human or murine OX40 at a K of about 90pM or less _D Binding to human or murine OX40,At a K of about 80pM or less _D Binds to human or murine OX40 at a K of about 70pM or less _D Binds to human or murine OX40 at a K of about 60pM or less _D Binds to human or murine OX40 at a K of about 50pM or less _D Binds to human or murine OX40 at a K of about 40pM or less _D Binding to human or murine OX40 or at a K of about 30pM or less _D OX40 agonists that bind to human or murine OX 40.

In some embodiments, the compositions, processes, and methods comprise at about 7.5×10 ⁵ K of 1/M.s or more _{Association with} Binds to human or murine OX40 at about 7.5X10 ⁵ K of 1/M.s or more _{Association with} Binds to human or murine OX40 at about 8X 10 ⁵ K of 1/M.s or more _{Association with} Binds to human or murine OX40 at about 8.5X10 ⁵ K of 1/M.s or more _{Association with} Binds to human or murine OX40 at about 9X 10 ⁵ K of 1/M.s or more _{Association with} Binds to human or murine OX40 at about 9.5X10 ⁵ K of 1/M.s or more _{Association with} Binds to human or murine OX40 or at about 1X 10 ⁶ K of 1/M.s or more _{Association with} OX40 agonists that bind to human or murine OX 40.

In some embodiments, the compositions, processes, and methods comprise at least about 2 x 10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.1X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.2X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.3X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.4X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.5X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.6X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 or at about 2.7X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.8X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 at about 2.9X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human or murine OX40 or at about 3X 10 ^-5 K of 1/s or less _{Dissociation of} OX40 agonists that bind to human or murine OX 40.

In some embodiments, the compositions, processes, and methods comprise an IC of about 10nM or less ₅₀ Binds to human or murine OX40 with an IC of about 9nM or less ₅₀ Binds to human or murine OX40 with an IC of about 8nM or less ₅₀ Binds to human or murine OX40 with an IC of about 7nM or less ₅₀ Binds to human or murine OX40 with an IC of about 6nM or less ₅₀ Binds to human or murine OX40 with an IC of about 5nM or less ₅₀ Binds to human or murine OX40 with an IC of about 4nM or less ₅₀ Binds to human or murine OX40 with an IC of about 3nM or less ₅₀ Binds to human or murine OX40 with an IC of about 2nM or less ₅₀ Binds to human or murine OX40 or has an IC of about 1nM or less ₅₀ OX40 agonists that bind to human or murine OX 40.

In some embodiments, the OX40 agonist is tamsulosin, also known as MEDI0562 or MEDI-0562. Tavolimab is available from the subsidiary company MedImmune of AstraZeneca, inc. Tavliximab is an immunoglobulin G1-kappa antibody [ Chile TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD 134)]Humanized and chimeric monoclonal antibodies. The amino acid sequence of the tamsulosin is shown in table 12. The tevoliximab comprises: n-glycosylation sites at positions 301 and 301', attachment of fucosylated complex bicontennary CHO-type glycans; in positions 22-95 (V) _H -V _L )、148-204(C _H 1-C _L )、265-325(C _H 2) And 371-429 (C) _H 3) (and the disulfide bonds within the heavy chain at positions 22"-95", 148"-204", 265"-325" and 371 "-429"); in positions 23'-88' (V) _H -V _L ) And 134'-194' (C) _H 1-C _L ) (and light chain intra-chain disulfide bonds at positions 23 '"-88'" and 134 '"-194'"); interchain heavy-heavy chain disulfide bonds at positions 230-230 "and 233-233"; and interchain heavy-light chain disulfide bonds at 224-214 'and 224 "-214'". The current clinical trials of Tavliximab in a variety of solid tumor indications include the national institutes of health, clinicaltrilals gov identification number NCT02318394 and NCT02705482.

In one embodiment, the OX40 agonist comprises an amino acid sequence consisting of SEQ ID NO:87 and the heavy chain represented by SEQ ID NO: 88. In one embodiment, 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 one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 99% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 98% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:87 and SEQ ID NO:88 has at least 97% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:87 and SEQ ID NO:88 has at least 96% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:87 and SEQ ID NO:88, and a heavy chain and a light chain having at least 95% identity.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of tavoriximab. In one embodiment, the OX40 agonist heavy chain variable region (V _H ) Comprising SEQ ID NO:89, an OX40 agonist light chain variable region (V _L ) Comprising SEQ ID NO:90 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:89 and SEQ ID NO:90 has a V with at least 99% identity to the sequence shown in 90 _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:89 and SEQ ID NO:90 has a V with at least 98% identity to the sequence shown in 90 _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:89 and SEQ ID NO:90 has a V with at least 97% identity to the sequence shown in 90 _H And V _L A zone. In one embodiment, the OX40 agonistComprises each of the sequences as set forth in SEQ ID NO:89 and SEQ ID NO:90 has a V with at least 96% identity to the sequence shown in 90 _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:89 and SEQ ID NO:90 has a V with at least 95% identity to the sequence shown in 90 _H And V _L A zone. In one embodiment, the OX40 agonist comprises an scFv antibody comprising an amino acid sequence that is substantially identical to SEQ ID NO:89 and SEQ ID NO:90 has a V with at least 99% identity to the sequence shown in 90 _H And V _L A zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 91. SEQ ID NO:92 and SEQ ID NO:93 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 94. SEQ ID NO:95 and SEQ ID NO:96, and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug administration with reference to tavoriximab. In one embodiment, the 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, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, which is tavoriximab. 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 biologic product, which is tavoriximab. OX40 agonist antibodies may be licensed by pharmaceutical authorities such as the us FDA and/or EMA of the european union. In some embodiments, the biosimilar is a composition provided as 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, which is tevogliximab. In some embodiments, the biosimilar is a composition provided as 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, which is tevogliximab.

Table 12: amino acid sequences of OX40 agonist antibodies related to tavoriximab

In some embodiments, the OX40 agonist is 11D4, which is a fully human antibody available from Pfizer, inc. The preparation and properties of 11D4 are 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 in their entirety. The amino acid sequence of 11D4 is shown in table 13.

In one embodiment, the OX40 agonist comprises an amino acid sequence consisting of SEQ ID NO:97 and the heavy chain given by SEQ ID NO: 98. In one embodiment, 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 one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:97 and SEQ ID NO:98 have heavy and light chains with at least 99% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:97 and SEQ ID NO:98 have heavy and light chains with at least 98% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:97 and SEQ ID NO:98 have heavy and light chains with at least 97% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:97 and SEQ ID NO:98 have heavy and light chains with at least 96% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:97 and SEQ ID NO:98 have heavy and light chains with at least 95% identity.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of 11D 4. In one embodiment, the OX40 agonist heavy chain variable region (V _H ) Comprising SEQ ID NO:99, an OX40 agonist light chain variable region (V _L ) Comprising SEQ ID NO:100 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:99 and SEQ ID NO:100 has at least 99% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:99 and SEQ ID NO:100 has a V with at least 98% identity to the sequence shown in 100 _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:99 and SEQ ID NO:100 has at least 97% identity to V _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:99 and SEQ ID NO:100 has a V with at least 96% identity to the sequence shown in 100 _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:99 and SEQ ID NO:100 has at least 95% identity of V _H And V _L A zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 101. SEQ ID NO:102 and SEQ ID NO:103 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 104. SEQ ID NO:105 and SEQ ID NO:106 and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown in fig. 106.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug administration with reference to 11D4. In one embodiment, the 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, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, which 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, and the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, which is 11D4.OX40 agonist antibodies may be licensed by pharmaceutical authorities such as the us FDA and/or EMA of the european union. In some embodiments, the biosimilar is a composition provided as 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, which is 11D4. In some embodiments, the biosimilar is a composition provided as 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, which is 11D4.

Table 13: 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 Pfizer, inc. The preparation and nature 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 in their entirety. The amino acid sequence of 18D8 is shown in table 14.

In one embodiment, the OX40 agonist comprises the amino acid sequence of SEQ ID NO:107 and SEQ ID NO:108, and a light chain as given in 108. In one embodiment, 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 one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:107 and SEQ ID NO:108 have heavy and light chains with at least 99% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:107 and SEQ ID NO:108 have at least 98% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:107 and SEQ ID NO:108 have at least 97% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:107 and SEQ ID NO:108 have heavy and light chains with at least 96% identity. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:107 and SEQ ID NO:108 have heavy and light chains with at least 95% identity.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of 18D 8. In one embodiment, the OX40 agonist heavy chain variable region (V _H ) Comprising SEQ ID NO:109, OX40 agonist light chain variable region (V _L ) Comprising SEQ ID NO:110 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:109 and SEQ ID NO:110 has at least 99% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:109 and SEQ ID NO:110 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:109 and SEQ ID NO:110 has at least 97% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:109 and SEQ ID NO:110 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:109 and SEQ ID NO:110 has a V with at least 95% identity to the sequence shown in 110 _H And V _L A zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 111. SEQ ID NO:112 and SEQ ID NO:113 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 114. SEQ ID NO:115 and SEQ ID NO:116, and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug administration with reference to 18D8. In one embodiment, the 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, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, which 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 approved or application-approved OX40 agonist antibody, and the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, which is 18D8.OX40 agonist antibodies may be licensed by pharmaceutical authorities such as the us FDA and/or EMA of the european union. In some embodiments, a biological analog is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, which is 18D8. In some embodiments, a biological analog is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in the reference drug or reference biological product, which is 18D8.

Table 14: amino acid sequence of an OX40 agonist antibody associated with 18D8

In some embodiments, the OX40 agonist is Hu119-122, which is a humanized antibody available from 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 in their entirety. The amino acid sequences of Hu119-122 are shown in Table 15.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VR) of Hu 119-122. In one embodiment, the OX40 agonist heavy chain variable region (V _H ) Comprising SEQ ID NO:117, OX40 agonist light chain variable region (V _L ) Comprising SEQ ID NO:118 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:117 and SEQ ID NO:118 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:117 and SEQ ID NO:118 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:117 and SEQ ID NO:118 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:117 and SEQ ID NO:118 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:117 and SEQ ID NO:118 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 119. SEQ ID NO:120 and SEQ ID NO:121 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NOs: 122. SEQ ID NO:123 and SEQ ID NO:124 and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a regulatory agency of the pharmaceutical authority in reference to Hu119-122. In one embodiment, the biological analog monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having 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, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, which 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, and the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, which is Hu119-122.OX40 agonist antibodies may be licensed by pharmaceutical authorities such as the us FDA and/or EMA of the european union. In some embodiments, the biosimilar is a composition provided as 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, which is Hu119-122. In some embodiments, the biosimilar is a composition provided as 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, which is Hu119-122.

Table 15: amino acid sequence of OX40 agonist antibody related to Hu119-122

In some embodiments, the OX40 agonist is Hu106-222, which is a humanized antibody available from GlaxoSmithKline plc. 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 in their entirety. The amino acid sequences of Hu106-222 are shown in Table 16.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VR) of Hu 106-222. In one embodiment, the OX40 agonist heavy chain variable region (V _H ) Comprising SEQ ID NO:125, an OX40 agonist light chain variable region (V _L ) Comprising SEQ ID NO:126 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:125 and SEQ ID NO:126 has at least 99% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:125 and SEQ ID NO:126 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:125 and SEQ ID NO:126 has a V with at least 97% identity to the sequence shown in SEQ ID NO. 126 _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:125 and SEQ ID NO:126 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is each of SEQ ID NO:125 and SEQ ID NO:126 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 127. SEQ ID NO:128 and SEQ ID NO:129 and CDR1, CDR2 and CDR3 domains and conservative amino acid substitutions thereof having the sequences set forth in SEQ ID NOs: 130. SEQ ID NO:131 and SEQ ID NO:132, and conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug administration with reference to Hu106-222. In one embodiment, the biological analog monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having 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, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, which 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 approved or application-approved OX40 agonist antibody, and the OX40 agonist antibody is provided in a different formulation than the formulation of the reference drug or reference biological product, which is Hu106-222.OX40 agonist antibodies may be licensed by pharmaceutical authorities such as the us FDA and/or EMA of the european union. In some embodiments, the biosimilar is a composition provided as 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, which is Hu106-222. In some embodiments, the biosimilar is a composition provided as 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, which is Hu106-222.

Table 16: amino acid sequence of OX40 agonist antibodies related to Hu106-222

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B 12). MEDI6469 is a murine monoclonal antibody. Weinberg et al, J.Immunother.2006,29,575-585. In some embodiments, the OX40 agonist is an antibody raised against a 9B12 hybridoma (registered by Biovest inc. (Malvern, MA, USA)), which is described in Weinberg et al, j.immunother.2006,29,575-585, the disclosure of which is hereby incorporated 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 one embodiment, the OX40 agonist is L106BD (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 one embodiment, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog 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 one embodiment, the OX40 agonist is a murine monoclonal antibody anti-mCD 134/mxx 40 (clone OX 86), which is commercially available from InVivoMAb, bioXcell Inc (parisonin, new hampshire, usa).

In one embodiment, the OX40 agonist is selected from the group consisting of those described in international patent application publication nos. 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. US 2010/136030, US 2014/377284, US 2015/190506 and US 2015/132088 (including 20E5 and 12H3 clones); and OX40 agonists in 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 respective disclosures of which are incorporated herein by reference in their entirety.

In one embodiment, the OX40 agonist is an OX40 agonist fusion protein or fragment, derivative, conjugate, variant or biological analog thereof as depicted by structure I-a (C-terminal Fc-antibody fragment fusion protein) or structure I-B (N-terminal Fc-antibody fragment fusion protein). The nature of structures I-A and I-B is 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 in their entirety. The amino acid sequence of the polypeptide domain of structure I-A is shown in FIG. 18, see Table 9. The Fc domain preferably comprises the complete constant domain (amino acids 17 to 230 of SEQ ID NO: 62), the complete hinge domain (amino acids 1 to 16 of SEQ ID NO: 62) or a portion of the hinge domain (e.g., amino acids 4 to 16 of SEQ ID NO: 62). Preferred linkers for linking the C-terminal Fc antibody may be selected from the group consisting of SEQ ID NOs: 63 to SEQ ID NO:72 includes linkers suitable for fusing additional polypeptides. Similarly, the amino acid sequence of the polypeptide domain of structure I-B is given in FIG. 18, see Table 10. If an Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein as in structure I-B, the sequence of the Fc module is preferably shown in SEQ ID NO:73, the linker sequence is preferably selected from the group consisting of SEQ ID NO:74 to SEQ ID NO: 76.

In one embodiment, 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 tamsulosin, 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 set forth in table 17, any combination of the foregoing, and fragments, derivatives, conjugates, variants and biological analogs thereof.

In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises more than one OX40 binding domain, which OX40 binding domain comprises an OX40L sequence. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains comprising an OX40 binding domain according to SEQ ID NO:133, a sequence of seq id no. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises more than one OX40 binding domain, which OX40 binding domain comprises a soluble OX40L sequence. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains comprising an OX40 binding domain according to SEQ ID NO: 134. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains comprising an OX40 binding domain according to SEQ ID NO: 135.

In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains, the OX40 binding domains being comprising a sequence that is complementary to each of SEQ ID NOs: 89 and SEQ ID NO:90 has a V with at least 95% identity to the sequence shown in 90 _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains, the OX40 binding domains being comprising a sequence that is complementary to each of SEQ ID NOs: 99 and SEQ ID NO:100 has at least 95% identity of V _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains, the OX40 binding domains being comprising a sequence that is complementary to each of SEQ ID NOs: 109 and SEQ ID NO:110 has a V with at least 95% identity to the sequence shown in 110 _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains, the OX40 binding domains being comprising a sequence that is complementary to each of SEQ ID NOs: 127 and SEQ ID NO:128 has at least 95% identity to V _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-a or I-B comprises one or more OX40 binding domains, the OX40 binding domains being comprising a sequence that is complementary to each of SEQ ID NOs: 125 and SEQ ID NO:126 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40 binding domains, the OX40 binding domain comprising a sequence of V each as set forth in Table 17 _H And V _L V having at least 95% identity to the sequence _H And V _L scFv domain of region, V _H And V _L The domains are connected by a linker.

Table 17: additional polypeptide domains useful as OX40 binding domains in fusion proteins (e.g., structures I-A and I-B) or scFv OX40 agonist antibodies

In one embodiment, 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 additional domains at the N-and/or C-terminus that are Fab or Fc fragment domains. In one embodiment, 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 additional domains at the N-and/or C-terminus that are Fab or Fc fragment domains, each of the soluble OX40 binding domains lacking a stem region that promotes trimerization and provides some distance from the cell membrane, but not a portion of the OX40 binding domain, the first and second peptide linkers independently having a length of 3 to 8 amino acids.

In one embodiment, 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 domain, each of the soluble TNF superfamily cytokine domains lacking a stem region, the first and second peptide linkers independently having a length of 3 to 8 amino acids, the TNF superfamily cytokine domain being an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383.MEDI6383 is an OX40 agonist fusion protein that can be prepared as described in U.S. patent No. 6,312,700, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the OX40 agonist is an OX40 agonist scFv antibody comprising any of the foregoing V _H The domain is linked to any of the foregoing V _L A domain.

In one embodiment, the OX40 agonist is the OX40 agonist monoclonal antibody MOM-1855 of Creative Biolabs, available from Creative Biolabs, inc (Shirley, new york, usa).

In one embodiment, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, available from BioLegend, inc (san diego, california, usa).

Akt inhibitors and DNA hypomethylators

In one embodiment, the first expansion and/or the rapid second expansion cell culture medium of the 2 nd or 3 rd generation processes or other processes described herein comprises an AKT inhibitor. The use of AKT inhibitors in TIL, MIL and PBL amplification procedures is described in International patent publication No. WO 2020/096927 A1, the disclosure of which is incorporated herein by reference in its entirety. AKT inhibitors disclosed herein may be used in connection with CCR and chemokine receptors disclosed herein in situations associated with the processes disclosed herein, or may be used alone with processes disclosed herein (e.g., the 2 nd or 3 rd generation processes) without CCR or chemokine receptor modification.

Suitable AKT inhibitors include AKT1, AKT2 and/or AKT3 inhibitors. In some embodiments, the AKT inhibitor is afuresertib (afuresertib) or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof, and combinations thereof. In some embodiments, the AKT inhibitor is eparatadine or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal or prodrug thereof, and combinations thereof. In some embodiments, the AKT inhibitor is selected from the following: aforotidine, aplatin (uprosisertib), eparatadine, cloth Lu Saiti cloth (borussertib), kava-sertib, mi Lanti ni (miranssertib), colestolide (oridon), vitamin Fu Lisai, AT7867, AT13148, BAY1125976, GSK-690693, MK-2206, LY294002, PF-04691502, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal or prodrug thereof, and combinations thereof. In some embodiments, the AKT inhibitor is added to the culture medium during the REP prophase of the 2 nd generation process, e.g., immediately after the addition of the fragmented or digested tumor to the culture. In some embodiments, the AKT inhibitor is added to the culture medium during the REP phase of the 2 nd generation process. In some embodiments, the AKT inhibitor is added to the culture medium during the initial phase of the 3 rd generation process. In some embodiments, the AKT inhibitor is added to the culture medium during the REP phase of the 3 rd generation process. In some embodiments, the AKT inhibitor is added to the culture medium during the REP phase of the TIL, MILs, or PBL manufacturing process. In some embodiments, the AKT inhibitor is added to the culture medium during TIL amplification. In some embodiments, the AKT inhibitor is added to the culture medium during the TIL amplification process comprising the genetic modification steps described herein. In some embodiments, the AKT inhibitor is added to the culture medium during a TIL amplification process comprising a transduction step of CCR or chemokine receptors. In some embodiments, the AKT inhibitor is an allosteric AKT inhibitor. In some embodiments, the AKT inhibitor is a covalent AKT inhibitor.

In some embodiments, use of AKT inhibitors during TIL amplification results in CD39 with differentiation ^- CD69 ^- TIL of the cells. In some embodiments, use of an AKT inhibitor during TIL amplification results in a CD39 having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (i.e., multiplied) relative to TIL prepared without the AKT inhibitor, e.g., using a 2 nd or 3 rd generation process modified to express CCR or chemokine receptors ^- CD69 ^- Cell amount TIL. In some embodiments, use of an AKT inhibitor during TIL amplification results in a dna having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% >,90% or 100% (i.e., multiplied) of IFNγ ⁺ TNFα ⁺ CD8 ⁺ T cell TIL.

In one embodiment, the invention includes a therapeutic TIL composition comprising a TIL comprising a CCR or chemokine receptor, the TIL being further optionally modified to stabilize or temporarily reduce expression of a protein (e.g., PD-1) by gene knock-out or gene knock-down of a gene (e.g., PDCD 1), and/or the TIL being prepared as an AKT inhibitor and exhibiting CD39 relative to a TIL prepared without the AKT inhibitor ^- CD69 ^- The amount of cells increases.

In one embodiment, the first expansion and/or the rapid second expansion cell culture medium of the 2 nd or 3 rd generation processes or other processes described herein comprises decitabine or a salt, co-crystal, solvate or hydrate thereof alone or in addition to an AKT inhibitor and alone or in combination with the CCR and chemokine receptors disclosed herein.

C. Alternative cell viability assays

Alternatively, the cell viability assay may be performed after the initial first amplification (sometimes referred to as initial subject amplification) using standard assays known in the art. Thus, in certain embodiments, the method comprises performing a cell viability assay after the initial first expansion. For example, trypan blue exclusion assays can be performed on samples of bulk TIL that selectively mark dead cells and allow viability assessment. Other assays for testing viability may include, but are not limited to, the alma blue assay and the MTT assay.

1. Cell counting, viability, flow cytometry

In some embodiments, cell count and/or viability is measured. Expression of markers (e.g., without limitation, CD3, CD4, CD8, and CD56, and any other disclosed or described herein) may be performed by flow cytometry using FACSCanto ^TM Flow cytometry (BD Biosciences) is measured with antibodies such as, but not limited to, those available from BD Biosciences (san jose, california). Cells can be manually counted using a disposable c-wafer hemocytometer (VWR, badavia, illinois), viability can be packaged using any method known in the art Including but not limited to trypan blue staining evaluation. Cell viability may also be determined based on U.S. patent application publication No. 2018/0282694, which is incorporated herein by reference in its entirety. Cell viability may also be determined based on U.S. patent application publication No. 2018/0280436 or international patent application publication No. WO/2018/081473, both of which are incorporated herein in their entirety for all purposes.

In certain instances, the subject TIL population can be immediately cryopreserved using the protocol discussed below. Alternatively, the subject TIL population may be REP-subjected and then cryopreserved as discussed below. Similarly, in situations where a genetically modified TIL would be used in therapy, the subject or population of REP TILs may be genetically modified for appropriate treatment.

2. Cell culture

In one embodiment, a method for amplifying TIL (including those discussed above and particularly illustrated in fig. 1 and 8, e.g., fig. 8B and/or 8C) 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 a 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 one embodiment, no more than one cell culture medium is used to amplify the amount of TIL. 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 (Invitrogen, calif. Bard). In this regard, the methods of the present invention advantageously reduce the amount of medium and the number of medium types required to amplify the amount of TIL. In one embodiment, the number of expanded TILs may comprise feeding the cells no more frequently than once every three or four days. Expanding cell numbers in a gas permeable container simplifies the procedure required to expand cell numbers by reducing the frequency of feeding required to expand cells.

In one embodiment, 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 one embodiment, the cell culture medium in the first and/or second gas permeable container lacks β -mercaptoethanol (BME).

In one embodiment, the duration of the method comprises: obtaining a tumor tissue sample from a mammal; 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 initial first amplification, or about 8 days as initial first amplification); transferring the TIL to a second gas permeable container, wherein the second gas permeable container contains a cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3, during expansion of the TIL 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 one embodiment, the duration of the method comprises: obtaining a tumor tissue sample from a mammal; 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 initial first expansion); transferring the TIL to a second permeable container, wherein the second permeable container contains a cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3, during a period of time in which the amount of TIL is expanded in the second permeable container for about 7 to 14 days or about 7 to 9 days (e.g., about 7 days, about 8 days or about 9 days, about 10 days or about 11 days).

In one embodiment, the duration of the method comprises: obtaining a tumor tissue sample from a mammal; as an initial first expansion, 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); 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, during a period of time in which the amount of TIL is expanded in the second gas permeable container for about 7 to 11 days (e.g., about 7 days, about 8 days, about 9 days, about 10 or about 11 days).

In one embodiment, 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. 2005/0106717 A1, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the TIL is amplified in a gas permeable bag. In one embodiment, the TIL is expanded using a cell expansion system that expands the TIL in a gas permeable bag, such as the Xuri cell expansion system W25 (GE Healthcare). In one embodiment, the TIL is expanded using a cell expansion system that expands the TIL in a gas permeable bag, such as a WAVE bioreactor system, also known as the Xuri cell expansion system W5 (GE Healthcare). In one embodiment, 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 one embodiment, TIL may be amplified in a G-Rex flask (commercially available from Wilson Wolf Manufacturing). Such embodiments allow cell populations from about 5 x 10 ⁵ Individual cells/cm ² Amplified to 10X 10 ⁶ Up to 30X 10 ⁶ Individual cells/cm ² . In one embodiment, this is not fed. In one embodiment, this is not done as long as the medium in the G-Rex flask is at a height of about 10 cm. In one embodiment, feeding is not performed, but more than one cytokine is added. In one embodiment, the cytokine may be added as a bolus, without mixing the cytokine with the medium. Such containers, devices and methods are known in the art and have been used to amplify TIL and include those described in U.S. patent application publication No. US 2014/0377739A1, international patent publication No. WO 2014/210036 A1, U.S. patent application publication No. US 2013/0112017 A1, international patent publication No. WO 2013/188427 A1, U.S. patent application publication No. US 2011/0137628 A1, U.S. patent No. US 2011/0136528 A18,809,050 B2, international patent publication No. WO 2011/072088 A2, U.S. patent application publication No. US 2016/0208216 A1, U.S. patent application publication No. US 2012/0244233 A1, international patent publication No. WO 2012/129201 A1, U.S. patent application publication No. US 2013/0102075 A1, U.S. patent No. US 8,956,860 B2, international patent publication No. WO 2013/173835 A1, U.S. patent application publication No. US 2015/0175966 A1, the disclosures of which are incorporated herein by reference in their entirety. Such processes are also described in Jin et al, J.Immunotherapy,2012,35:283-292.

Selectable knockdown or knock-out in TIL

In some embodiments, the amplified TILs of the present invention are further manipulated to alter protein expression in a transient manner prior to, during, or after the amplification step, including during a closed, sterile manufacturing process (each provided herein). In some embodiments, the temporarily altered protein expression is due to temporary gene editing. In some embodiments, the amplified TIL of the invention is treated with Transcription Factors (TF) and/or other molecules capable of temporarily altering the expression of proteins in the TIL. In some embodiments, the TF and/or other molecules that transiently alter protein expression provide for altered tumor antigen expression and/or alter the 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 into a TIL population, such as by ribonucleic acid (RNA) insertion, including insertion of messenger RNAs (mrnas) or small (or short) interfering RNAs (sirnas), 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 TIL of the present invention undergoes a temporal change in protein expression. In some embodiments, the transient change in protein expression occurs in the subject TIL population prior to the first amplification. In some embodiments, the temporary change in protein expression occurs after the first amplification. In some embodiments, the transient change in protein expression occurs in the subject TIL population prior to the second amplification. In some embodiments, the temporary change in protein expression occurs after the second amplification.

In one embodiment, 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 Tsong, biophys.j.1991,60,297-306 and U.S. patent application publication No. 2014/0227237 A1, the respective disclosures of which are incorporated herein by reference in their entirety. In one embodiment, 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 Graham and van der Eb, virology 1973,52,456-467; wigler et al, proc.Natl.Acad.Sci.1979,76,1373-1376, chen and Okayarea, 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 their entirety. In one embodiment, the method of transiently altering protein expression in a TIL population comprises the step of lipofection. Liposome transfection methods such as 1 using the cationic lipids N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleylphospholipid ethanolamine (DOPE) in filtered water: methods of 1 (w/w) liposome formulation are known in the art, described in Rose et al, biotechniques 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA,1987,84,7413-7417 and U.S. Pat. No. 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 their entirety. In one embodiment, a method of transiently altering protein expression in a TIL population comprises using us patent 5,766,902;6,025,337;6,410,517; a transfection step of the methods described in 6,475,994 and 7,189,705; the respective disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, TILs of the invention (including TILs modified to express CCR) are further modified to temporarily or permanently inhibit expression of one or more genes using the methods described in international patent application nos. WO 2019/136456 A1 or WO 2019/210131 A1 (each of which is incorporated herein by reference in its entirety), including methods in which gene editing of a TIL to knock out a particular gene of interest (e.g., genes encoding PD-1 and CTLA-4) is described.

In some embodiments, the temporary change in protein expression results in an increase in stem memory T cells (TSCM). TSCM is an early progenitor cell for antigen to undergo central memory T cells. TSCM generally exhibits the ability to define long-term survival, self-regeneration, and pluripotency of stem cells, and is generally required to produce effective TIL products. TSCM showed enhanced antitumor activity in a mouse model of adoptive cell transfer compared to other T cell subsets. In some embodiments, the transient alteration in protein expression results in a population of TILs having a composition comprising a high proportion of TSCM. In some embodiments, the temporary change in 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, the transient alteration in 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, the temporary alteration in 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, the temporal change in 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, the temporary change in protein expression results in the antigen undergoing T cell resuscitation. In some embodiments, resuscitation includes, for example, increasing proliferation, increasing T cell activation, and/or increasing antigen recognition.

In some embodiments, a temporary change in protein expression alters expression of a large fraction of T cells to preserve a tumor derived TCR reservoir. In some embodiments, the transient alteration in protein expression does not alter the tumor-derived TCR repertoire. In some embodiments, the transient alteration in protein expression maintains a tumor-derived TCR reservoir.

In some embodiments, the temporal change in the protein results in a change in the expression of a particular gene. In some embodiments, the temporal altered targeting of protein expression includes, but is not limited to, the following genes: PD-1 (also known as PDCD1 or CC 279), TGFBR2, CCR4/5, CBL-B (also known as CBLB and Cbl-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM-3, LAG-3, TIGIT, TGF beta, CCR2, CCR4, CCR5, CXCR1, CXCR 3, CCL2 (MCP-1), CCL3 (MIP-1 alpha), CCL4 (MIP 1-beta), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte select-associated High Mobility Group (HMG) boxes (TOX), ankyrin repeat domain 11 (ANKRD 11), BCL6 repressor (BCOR), and/or Protein Kinase A (PKA). In some embodiments, the transient alteration in protein expression targets a gene selected from the group consisting of: PD-1, TGFBR2, CCR4/5, CBL-B, CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM-3, LAG-3, TIGIT, TGF-beta, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP 1- β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, thymocyte selection-related High Mobility Group (HMG) cassette (TOX), ankyrin repeat domain 11 (ANKRD 11), BCL6 co-inhibitor (BCOR) and/or cAMP Protein Kinase A (PKA). In some embodiments, the transient alteration in protein expression targets PD-1. In some embodiments, the transient alteration in protein expression targets TGFBR2. In some embodiments, a transient change in protein expression targets CCR4/5. In some embodiments, a transient alteration in protein expression targets CBL-B. In some embodiments, the temporary alteration in protein expression targets CISH. In some embodiments, the transient alteration in protein expression targets CCR (chimeric co-stimulatory receptor). In some embodiments, the transient alteration in protein expression targets IL-2. In some embodiments, the temporary change in protein expression targets IL-12. In some embodiments, the transient alteration in protein expression targets IL-15. In some embodiments, the transient alteration in protein expression targets IL-21. In some embodiments, a transient change in protein expression targets NOTCH 1/2ICD. For some embodiments, a transient change in protein expression targets TIM-3. In some embodiments, a transient change in protein expression targets LAG-3. In some embodiments, the transient alteration in protein expression targets TIGIT. In some embodiments, the transient alteration in protein expression targets tgfβ. In some embodiments, the transient alteration in protein expression targets CCR1. In some embodiments, the transient alteration in protein expression targets CCR2. In some embodiments, the transient alteration in protein expression targets CCR4. In some embodiments, the transient alteration in protein expression targets CCR5. In some embodiments, a transient alteration in protein expression targets CXCR1. In some embodiments, a transient alteration in protein expression targets CXCR2. In some embodiments, a transient change in protein expression targets CSCR3. In some embodiments, the transient alteration in protein expression targets CCL2 (MCP-1). In some embodiments, the transient alteration in protein expression targets CCL3 (MIP-1α). In some embodiments, the transient alteration in protein expression targets CCL4 (MIP 1- β). In some embodiments, the transient alteration in protein expression targets CCL5 (RANTES). In some embodiments, the transient alteration in protein expression targets CXCL1. In some embodiments, a transient change in protein expression targets CXCL8. In some embodiments, the transient alteration in protein expression targets CCL22. In some embodiments, the transient alteration in protein expression targets CCL17. In some embodiments, the transient alteration in protein expression targets VHL. In some embodiments, the transient alteration in protein expression targets CD44. In some embodiments, the transient alteration in protein expression targets PIK3CD. In some embodiments, the transient alteration in protein expression targets SOCS1. In some embodiments, the transient alteration in protein expression targets thymic cells to 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 in protein expression targets a BCL6 co-repressor (BCOR). In some embodiments, the transient alteration in protein expression targets cAMP Protein Kinase A (PKA).

In some embodiments, the transient alteration in protein expression results in increased and/or over-expression of the chemokine receptor. In some embodiments, chemokine receptors that are overexpressed due to transient protein expression include receptors for ligands including, but not limited to, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP 1- β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL 17.

In some embodiments, the transient alteration in protein expression results in reduced and/or decreased expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2 and/or TGFβ (including resulting in, for example, a TGFβ pathway block). In some embodiments, the temporary change in protein expression results in reduced and/or decreased expression of CBL-B. In some embodiments, the temporary change in protein expression results in reduced and/or decreased expression of CISH.

In some embodiments, the transient alteration in protein expression results in increased and/or over-expression of chemokine receptors, for example, to improve TIL movement or movement to the tumor site. In some embodiments, the transient alteration in protein expression results in increased CCR (chimeric co-stimulatory receptor) and/or overexpression. In some embodiments, the transient alteration in protein expression results in increased and/or overexpression of a chemokine receptor selected from CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR 3.

In some embodiments, the temporary change in protein expression results in increased and/or over-expression of interleukins (including membrane-bound interleukins). In some embodiments, the transient alteration in protein expression results in increased and/or over-expression of an interleukin selected from the group consisting of IL-2, IL-12, IL-15, and/or IL-21. For example, in some embodiments, electroporation of membrane-bound IL-2, IL-12, IL-15, and/or IL-21 (mbiL-2, mbiL-12, mbiL-15, and/or mbiL-21, and single chain variants such as single chain mbiL-12) may be included in the TILs of the invention, alone or in combination with the CCR and chemokine receptors described herein, and alone or in combination with gene knockdown or gene knock-out of the genes described herein. Compositions and methods related to the foregoing are described herein and in Zhang et al, j. Immunother. Cancer 2020,8,000210, international patent publication No. WO 2020/123716 A1 and U.S. patent application publication No. US 2017/0291934 A1, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, a transient change in protein expression results in increased and/or over-expression of NOTCH 1/2 ICD. In some embodiments, the transient alteration in protein expression results in increased and/or over-expression of VHL. In some embodiments, the temporary change in protein expression results in increased and/or over-expression of CD 44. In some embodiments, the temporary change in protein expression results in increased and/or over-expression of PIK3 CD. In some embodiments, the temporary change in protein expression results in increased and/or over-expression of SOCS 1. In some embodiments, the temporary alteration in protein expression results in increased and/or over-expression of CD40 ligand (CD 40L). In some embodiments, the temporary change in protein expression results in increased and/or over-expression of cAMP Protein Kinase A (PKA). In some embodiments, the temporary change in protein expression results in a decrease and/or decrease in expression of cAMP Protein Kinase A (PKA).

In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in expression of two molecules selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in the expression of PD-1 and a molecule selected from the group consisting of LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (Br 3), and combinations thereof. In some embodiments, the temporal change in protein expression results in reduced and/or decreased expression of PD-1, LAG-3, CISH, CBL-B, TIM-3, and combinations thereof. In some embodiments, the temporal change in protein expression results in a decrease and/or decrease in expression of one of PD-1 and LAG-3, CISH, CBL-B, TIM-3, and combinations thereof. In some embodiments, the temporal change in protein expression results in reduced and/or decreased expression of PD-1 and LAG-3. In some embodiments, the temporary alteration in protein expression results in reduced and/or decreased expression of PD-1 and CISH. In some embodiments, the temporary alteration in protein expression results in reduced and/or decreased expression of PD-1 and CBL-B. In some embodiments, the temporal change in protein expression results in reduced and/or decreased expression of LAG-3 and CISH. In some embodiments, the temporal change in protein expression results in a decrease and/or decrease in the expression of LAG-3 and CBL-B. In some embodiments, the temporary alteration in protein expression results in reduced and/or decreased expression of CISH and CBL-B. In some embodiments, a temporary change in protein expression results in a decrease and/or decrease in expression of TIM-3 and PD-1. In some embodiments, the temporary alteration of protein expression results in a decrease and/or decrease in the expression of TIM-3 and LAG-3. In some embodiments, the temporary alteration in protein expression results in reduced and/or decreased expression of TIM-3 and CISH. In some embodiments, the temporary alteration of protein expression results in a decrease and/or decrease in the expression of TIM-3 and CBL-B.

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, the temporal change in protein expression results in a decrease and/or decrease in expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3) and combinations thereof, and an increase and/or enhancement in expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH, CBL-B, and combinations thereof, and an increase and/or enhancement in expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, TIL is a gene modified further to target stable or transient changes in protein expression including, but not limited to, CD38, HPK1, YAP1, PTPN22, CBL-B, PGC1 a, NT-pgc1a, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, CD40L, and/or c-Jun. In some embodiments, TIL is a gene selected from the group consisting of CD38, HPK1, YAP1, PTPN22, CBL-B, PGC1 a, NT-PGC1 a, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, CD40L, c-Jun, and combinations thereof, which is further genetically modified to target an alteration in protein expression.

In some embodiments, the transient alteration in protein expression targets CD38. In some embodiments, the transient alteration in protein expression targets HPK1. In some embodiments, the transient alteration in protein expression targets YAP1. In some embodiments, a transient change in protein expression targets PTPN22. In some embodiments, a transient alteration in protein expression targets CBL-B. In some embodiments, the transient alteration in protein expression targets pgc1α. In some embodiments, a transient alteration in protein expression targets NT-PGC1 alpha. In some embodiments, a transient alteration in protein expression targets CXCR1. In some embodiments, a transient alteration in protein expression targets CXCR2. In some embodiments, a transient alteration in protein expression targets CXCR3. In some embodiments, a transient alteration in protein expression targets CXCR4. In some embodiments, a transient alteration in protein expression targets CXCR5. In some embodiments, a transient alteration in protein expression targets CXCR6. In some embodiments, the transient alteration in protein expression targets CX3CR1. In some embodiments, the transient alteration in protein expression targets CCR1. In some embodiments, the transient alteration in protein expression targets CCR2. In some embodiments, the transient alteration in protein expression targets CCR4. In some embodiments, the transient alteration in protein expression targets CCR5. In some embodiments, the transient alteration in protein expression targets CCR6. In some embodiments, the transient alteration in protein expression targets CCR7. In some embodiments, the transient alteration in protein expression targets CCR8. In some embodiments, the transient alteration in protein expression targets CCR9. In some embodiments, the transient alteration in protein expression targets CCR10. In some embodiments, the transient alteration in protein expression targets the bat. In some embodiments, the transient alteration in protein expression targets c-Jun. In some embodiments, the transient alteration in protein expression targets CD40L.

In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in expression of PD-1 and one or more molecules selected from CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in the expression of PD-1 and two or more molecules selected from CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in the expression of PD-1 and three or more molecules selected from CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and/or decrease in expression of PD-1 and four or more molecules selected from CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporary alteration in protein expression results in reduced and/or decreased expression of CD38, HPK1, and/or YAP 1. In some embodiments, the temporary alteration in protein expression results in a decrease and/or decrease in expression of CD38 and a decrease and/or decrease in expression of PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in reduced and/or decreased expression of HPK1 and reduced and/or decreased expression of PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in reduced and/or decreased expression of YAP1 and reduced and/or decreased expression of PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in a decrease and/or decrease in expression of PTPN22 and a decrease and/or decrease in expression of CD38, HPK1, YAP1, and combinations thereof. In some embodiments, the temporal change in protein expression results in a decrease and/or decrease in expression of CBL-B and a decrease and/or decrease in expression of CD38, HPK1, YAP1, and combinations thereof.

In some embodiments, the transient alteration in protein expression results in the increase and/or overexpression of one or more molecules selected from pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the transient alteration in protein expression results in increased and/or overexpression of two or more molecules selected from pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the transient alteration in protein expression results in increased and/or overexpression of three or more molecules selected from pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the transient alteration in protein expression results in increased and/or overexpression of four or more molecules selected from pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the transient alteration in protein expression results in the increase and/or overexpression of five or more molecules selected from pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof.

In some embodiments, the temporal alteration in protein expression results in a decrease and/or decrease in expression of a molecule selected from the group consisting of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof and an increase and/or enhancement in expression of pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the temporal alteration in protein expression results in a decrease and/or decrease in expression of CD38 and an increase and/or enhancement in expression of pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the temporal alteration in protein expression results in a decrease and/or decrease in expression of HPK1 and an increase and/or enhancement in expression of pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the temporal alteration in protein expression results in a decrease and/or decrease in expression of YAP1 and an increase and/or enhancement in expression of pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the temporal alteration in protein expression results in a decrease and/or decrease in expression of PTPN22 and an increase and/or enhancement in expression of pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof. In some embodiments, the temporal alteration in protein expression results in a decrease and/or decrease in expression of CBL-B and an increase and/or enhancement in expression of pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof.

In some embodiments, the transient alteration in protein expression results in an increase and/or enhancement in expression of a molecule selected from the group consisting of pgc1α, NT-pgc1α, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, c-Jun, and combinations thereof and a decrease and/or reduction in expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of PGC1alpha and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of NT-PGC 1alpha and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CXCR1 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CXCR2 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CXCR3 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CXCR4 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CXCR5 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CXCR6 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CX3CR1 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR1 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR2 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR4 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR5 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR6 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR7 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR8 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR9 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of CCR10 and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of BATF and decreased and/or decreased expression of CD38, HPK1, YAP1, PTPN22, CBL-B, and combinations thereof. In some embodiments, the temporal change in protein expression results in increased and/or enhanced expression of c-Jun and decreased and/or reduced expression of CD38, HPK1, YAP1, PTPN22, CBL-B, 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, the temporary alteration of protein expression is induced by treating the TIL with a Transcription Factor (TF) and/or other molecules capable of temporarily altering protein expression in the TIL. In some embodiments, intracellular delivery of Transcription Factors (TF) and/or other molecules that temporarily alter protein expression is performed using a microfluidic platform without an SQZ vector. Such methods of demonstrating the ability to deliver proteins (including transcription factors) to a variety of primary human cells (including T cells) are described in U.S. patent application publication nos. US 2019/0093073 A1, US 2018/0201889 A1, and US 2019/0017072 A1, the respective disclosures of which are incorporated herein by reference in their entirety. Such methods may be employed by the present invention to expose a TIL population to Transcription Factors (TFs) and/or other molecules capable of inducing expression of a transient protein, wherein the TFs and/or other molecules capable of inducing expression of a transient protein provide for increased expression of a tumor antigen and/or increased numbers of tumor antigen-specific T cells in the TIL population, thereby resulting in increased therapeutic efficacy of the reprogrammed and reprogrammed TIL population compared to the non-reprogrammed TIL population. In some embodiments, reprogramming affects the initial or previous (i.e., prior to reprogramming) population of stress T cells and/or central memory T cell subpopulations relative to TILs as described herein.

In some embodiments, transcription Factors (TF) include, but are not limited to, TCF-1, NOTCH 1/2ICD, MYB, BATF, CD L and/or C-Jun. 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 bat f. In some embodiments, the Transcription Factor (TF) is c-Jun. In some embodiments, the Transcription Factor (TF) is CD40L. In some embodiments, the Transcription Factor (TF) is administered with an induced pluripotent stem cell culture (iPSC) such as commercial KNOCKOUT Serum Replacement (Gibco/thermo fisher) 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 temporarily altering protein expression as described above may be combined with methods of genetically modifying a population of TILs, such as gene editing for expression of CCR as described elsewhere herein, which includes the step of stably incorporating the 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 one embodiment, the method of genetically modifying a population of TILs comprises a retroviral transduction step. In one embodiment, the method of genetically modifying a TIL population comprises a lentiviral transduction step. Lentiviral transduction systems are known in the art and are described, for example, in Levine et al, proc.nat' l acad.sci.2006,103,17372-77; zufferey et al, 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 their entirety. In one embodiment, the method of genetically modifying a TIL population comprises a gamma retrovirus transduction step. Gamma retroviral transduction systems are known in the art and described, for example, in Cepko and Pear, cur.prot.mol.biol.1996,9.9.1-9.9.16, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, a method of genetically modifying a population of TILs comprises the step of transposon mediated gene transfer. Transposon mediated gene transfer systems are known in the art and include systems in which a 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 a transgenic cell, e.g., the transposase is provided as mRNA (e.g., mRNA comprising a cap and a polyadenylation tail). Suitable transposon mediated gene transfer systems include salmon-type Tel-like translocases (SB or sleeping beauty translocases) such as SB10, SB11 and SB100x, and engineered enzymes with increased enzymatic activity are described, for example, in Hackett et al, mol. Therapy 2010,18,674-83 and U.S. patent No. 6,489,458, the respective disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the temporary change in protein expression in the TIL is induced by a small interfering RNA (siRNA), sometimes referred to as short interfering RNA or silent RNA, which is a double stranded RNA molecule, typically 19 to 25 base pairs in length. siRNA is used for RNA interference (RNAi), which uses complementary nucleotide sequences to interfere with the expression of specific genes. siRNA can be used to temporarily knock down genes in TIL, also to modify CCR according to the present invention.

In some embodiments, the transient alteration of protein expression in the TIL is induced by self-delivered RNA interference (sdRNA), which is a chemical synthesis with a high percentage of 2' -OH substitutions (typically fluorine or-OCH) ₃ ) An asymmetric siRNA duplex comprising a 20 nucleotide antisense (guide) strand and a 13 to 15 base synonymous (passenger) strand and conjugated to cholesterol at its 3' end using a tetraethylene 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 for RNA interference (RNAi) that uses complementary nucleotide sequences to interfere with the expression of a particular gene. Sdrnas are covalently and hydrophobically modified RNAi compounds that enter cells without the need for a delivery vehicle. Sdrnas are substantially asymmetric chemically modified nucleic acid molecules with minimal double stranded regions. SdRNA molecules generally contain a single-stranded region and a double-stranded region, and in the single-stranded region and double-stranded region of the molecule The chain region may contain various chemical modifications. Furthermore, the sdRNA molecules can be attached to hydrophobic conjugates, such as known and advanced sterol-type molecules as described herein. Sdrnas and related methods of making such sdrnas are also widely described, for example, in U.S. patent application publication nos. US2016/0304873A1, US2019/0211337A1, US 2009/01331360 A1 and US2019/0048341A1, and U.S. patent nos. 10,633,654 and 10,913,948B2, the disclosures of each of which are incorporated herein by reference in their entirety. To optimize sdRNA structure, chemistry, targeting location, sequence preference, etc., algorithms were developed and applied to efficacy prediction of sdrnas. Based on these analyses, functional sdRNA sequences are generally defined as having a reduction in expression of more than 70% at a concentration of 1 μm, with a probability of more than 40%.

Double-stranded DNA (dsRNA) can generally be used to define any molecule comprising a pair of RNA complementary strands, typically synonymous (passenger) and antisense (guide) strands, and can include a single-stranded overhang region. With respect to siRNA, the term dsRNA generally refers to a precursor molecule comprising the sequence of an siRNA molecule that is released from a larger dsRNA molecule by the action of a cleaving enzyme system comprising Dicer enzymes.

In some embodiments, the method comprises a temporary change in protein expression of a TIL population, the TIL population comprising TIL modified to express CCR, comprising use of siRNA or sdRNA. Methods using siRNA and sdRNA have been described in Khvorova and Watts, nat. Biotechnol.2017,35,238-248; byrne et al, J.ocul. Pharmacol. Ther.2013,29,855-864; and Ligtenberg et al, mol. Therapy,2018,26,1482-93, the disclosures of which are incorporated herein by reference in their entirety. In one embodiment, the delivery of the siRNA is accomplished using electroporation or cell membrane disruption (e.g., extrusion or SQZ methods). In one embodiment, delivery of the sdrnas to the TIL population is not accomplished using electroporation, SQZ, or other methods, but rather the TIL population is exposed to sdrnas in culture 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 the 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 and 3 days. In one embodiment, delivering the sdRNA to the TIL population is accomplished using exposing the TIL population to sdRNA in a concentration of 10. Mu.M/10,000 TIL in the medium for a period of 1 to 3 days. In one embodiment, delivering the sdRNA to the TIL population is accomplished using exposing the TIL population to sdRNA in a concentration of 50. Mu.M/10,000 TIL in the medium for a period of 1 to 3 days. In one embodiment, delivering the sdRNA to the population of TILs is accomplished using exposing the population of TILs to the sdRNA in a medium at a concentration between 0.1. Mu.M/10,000 TILs and 50. Mu.M/10,000 TILs for a period of 1 to 3 days. In one embodiment, delivering the sdRNA to the TIL population is accomplished in culture medium by exposing the TIL population to a concentration of between 0.1 μm/10,000 TIL and 50 μm/10,000 of sdRNA during 1 to 3 days of use, wherein the exposing to the sdRNA is performed two, three, four, or five times by adding fresh sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. patent application publications US 2011/0039914 A1, US 2013/01331141 A1 and US 2013/013442 A1, and U.S. patent No. 9,080,171, the disclosures of which are incorporated herein by reference in their entirety.

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-4 TIM-3, LAG-3, TIGIT, TGF beta, TGFBR2, cAMP Protein Kinase A (PKA), BAFF BR3, CISH, CBL-B, CD38, HPK1, YAP1, and/or PTPN 22. In some embodiments, the reduced expression is determined based on a percentage of gene silencing, e.g., assessed 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 employed by the methods of the invention to successfully deliver sdRNA to TIL as described herein. The combination of modification of the backbone by asymmetric siRNA structures with hydrophobic ligands (see, e.g., ligtenberg et al, mol. Therapy,2018,26,1482-93 and U.S. patent application publication 2016/0304873 A1, the disclosures of which are incorporated herein by reference in their entirety) allows for the penetration of sdrnas through cultured mammalian cells by simple addition to the culture medium without additional formulation and methods, leveraging the nuclease stability of the sdrnas. This stability allows for supporting a constant level of RNAi-mediated reduction in target gene activity, only by maintaining an effective concentration of sdRNA in the medium. While not being limited by theory, backbone stabilization of the 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 occurs through various specific sirnas or sdrnas. In some embodiments, the siRNA or sdRNA containing several unmodified ribose residues is a fully modified sequence substitution 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 than 8 days. In some embodiments, the expression-reducing effect is reduced 10 days or more than 10 days after treatment of the TIL with the siRNA or sdRNA. In some embodiments, target expression is maintained with a reduction in expression of greater than 70%. In some embodiments, the target expression in the TIL is maintained with a reduction in expression of more than 70%. In some embodiments, reduced expression of the PD-1/PD-L1 pathway allows 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 due to siRNA or sdRNA results in increased TIL proliferation.

In some embodiments, the sdRNA sequences used in the present 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 present invention exhibit a 95% reduction in target gene expression. In some embodiments, the sdRNA sequences used in the present invention exhibit 99% reduction in target gene expression. 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 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 the 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 the 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 the 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 the 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 the 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 affect the effective 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 oligonucleotide is modified to include one or more hydrophobic modifications, 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' -O-ethyl), i.e., 2' -alkoxy, 2' -amino, 2' -S-alkyl, 2' -halo (including 2' -fluoro), T-methoxyethoxy, 2' -allyloxy (-OCH) ₂ CH＝CH ₂ ) 2 '-propynyl, 2' -propyl, ethynyl, ethenyl, propenyl, cyano and the like. In one embodiment, the sugar moiety may be a hexose and is incorporated into an oligonucleotide as described in Augustyns et al, nucleic acids res 1992,18,4711, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the double stranded siRNA or sdRNA oligonucleotides of the invention are double stranded in their full length, i.e., the molecules do not have an overhanging single stranded sequence at either end, i.e., are blunt ended. In some embodiments, the respective nucleic acid molecules may have different lengths. In other words, the double stranded siRNA or sdRNA oligonucleotides of the invention are not full length double stranded. For example, when two separate nucleic acid molecules are used, a first molecule, e.g., comprising an antisense sequence, may be longer than a second molecule that hybridizes to it (leaving a portion of the molecules single stranded). In some embodiments, when a single nucleic acid molecule is used, a portion of the molecule may remain single stranded at either end.

In some embodiments, the double stranded siRNA or sdRNA oligonucleotides of the invention contain mismatches and/or loops or bulges but are double stranded for at least about 70% of the length of the oligonucleotide. In some embodiments, the double-stranded oligonucleotides of the invention are double-stranded for at least about 80% of the length of the oligonucleotide. In another embodiment, the double stranded siRNA or sdRNA oligonucleotides of the invention are double stranded for at least about 90% to 95% of the length of the oligonucleotide. In some embodiments, the double stranded siRNA or sdRNA oligonucleotides of the invention are double stranded for at least about 96% to 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 free of nuclease action, for example, by modification such as 3 'or 5' linkage as described in U.S. patent No. 5,849,902, the disclosure of which is incorporated herein by reference in its entirety. For example, oligonucleotides can be made resistant by incorporating "blocking groups". The term "blocking group" as used herein refers to a moiety that can be attached to an oligonucleotide or a nucleomonomer as a synthetic protecting or coupling group (e.g., FITC, propyl (CH) ₂ -CH ₂ -CH ₃ ) Diols (-O-CH) ₂ -CH ₂ -O-) phosphate (PO ₃ ^2” ) Substituents of hydrogen phosphonates or phosphoramidites (for example in addition to OH groups). "blocking groups" may also include "end blocks" that protect the 5 'and 3' ends of the oligonucleotidesThe group "or" exonuclease blocking group "includes 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 can result in 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, 500 enhancing cellular uptake of siRNA or sdRNA. 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 a synonymous chain (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 double stranded region (10 to 15 bases long required for efficient RISC entry) and a single stranded region of 4 to 12 nucleotides long; having a 13 nucleotide double strand. 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 and does not interfere with RISC entry. In some embodiments, the pattern of chemical modification in the guide strand includes that a majority of the C and U nucleotides are 2'f modified and the 5' end is 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 molecular region is between 8 and 15 nucleotides in length. In some embodiments, the double-stranded molecular region is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long. In some embodiments, the double stranded region is 13 nucleotides long. There may be 100% complementarity between the guide and the passenger strand, or there may be more than one mismatch between the guide and the passenger strand. In some embodiments, on one end of the double-stranded molecule, the molecule is blunt-ended or has one nucleotide overhang. The single stranded region of the molecule is in some embodiments 4 to 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 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 reduced stability. In certain instances, 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 hours or more than 24 hours (including any intermediate value). In some embodiments, the siRNA or sd-RNA has a half-life in the medium of longer than 12 hours.

In some embodiments, the siRNA or sdRNA is optimized to increase efficacy and/or reduce toxicity. In some embodiments, the nucleotide length of the guide and/or passenger strand and/or the number of phosphorothioate modifications in the guide and/or passenger strand may affect the efficacy of the RNA molecule in some aspects, whereas substitution of the 2 '-fluoro (2' f) modification with the 2 '-O-methyl (2' ome) modification may affect the toxicity of the molecule in some aspects. In some embodiments, it is contemplated that reducing the 2' f content of the molecule may reduce the toxicity of the molecule. In some embodiments, the amount of phosphorothioate modification in the RNA molecule can affect the efficiency of molecular uptake, e.g., passive uptake, of the molecule into the cell. In some embodiments, the siRNA or sdRNA does not have a 2' f modification, but is still characterized by equal cell uptake and tissue penetration efficiency.

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 phosphate modified nucleotides. The guide strand may contain more than one modification that imparts increased stability and does not interfere with RISC entry. Phosphate modified nucleotides (e.g., phosphorothioate modified nucleotides) may be located at the 3 'end, the 5' end, or distributed throughout the guide strand. In some embodiments, the 10 nucleotides at 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 guide strand position 1 (the nucleotide at the 5 'most position of the guide strand) is modified and/or phosphorylated by 2' ome. The C and U nucleotides in the guide strand may be modified with 2' F. For example, the C and U nucleotides at positions 2 to 10 (or positions in the guide strand corresponding to different lengths) of the 19 nt guide strand 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 at positions 11 to 18 (or positions in the guide strand corresponding to different lengths) of the 19 nt guide strand may be modified with 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 are modified with 2'f and the 5' end of the guide strand is phosphorylated. In other embodiments, positions 1 and 11 to 18 are C or U modified with a 2'ome and the 5' end of the guide strand is phosphorylated. In other embodiments, positions 1 and C or U at positions 11 to 18 are 2' ome modified, the 5' end of the guide strand is phosphorylated and C or U at positions 2 to 10 is 2' f modified.

The 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 techniques. The ability to transfect difficult to transfect cell lines, high in vivo activity and ease of use are features of such compositions and methods, presenting significant functional advantages over traditional siRNA-based techniques, and thus employing the sdRNA approach in several embodiments of the methods for reduced expression of a target gene in the TIL 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 are available from advira LLC (mass, or the like).

The siRNA and sdRNA may be formed as hydrophobically modified siRNA-antisense oligonucleotide hybrid structures and are disclosed, for example, in Byrne et al, j.ocular pharmacol.therapeutic, 2013,29,855-864, the disclosure of which is incorporated herein by reference in its entirety.

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 oligonucleotides may be combined with a transmembrane delivery system for delivery to the cells. 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 the need for any delivery agent. In certain embodiments, the method comprises delivering the siRNA or sdRNA oligonucleotide to the TIL population using a transmembrane delivery system.

The oligonucleotide and oligonucleotide composition are contacted (e.g., contacted, also referred to herein as administered or delivered) with a TIL described herein and ingested, including by passive ingestion of the TIL. The sdRNA can be added to the TIL described herein during the first amplification (e.g., during 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 step E is collected, during or after step F is collected, before or during step F final formulation and/or transfer to an infusion bag, and before any optional cryopreservation step of step F. Alternatively, the sdRNA may be added after thawing from any of the cryopreservation steps of step F. In one embodiment, one or more sdrnas (which target genes as described herein including PD-1, LAG-3, TIM-3, CISH, CBL-B, CD38, HPK1, YAP1 and/or PTPN 22) may be added to a cell culture medium comprising TIL and other agents at a concentration selected from 100nM to 20mM, 200nM to 10mM, 500nM to 1mM, 1 μm to 100 μm and 1 μm to 100 μm. In one embodiment, one or more than one sdRNA targeted to genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBL-B, may be added to the cell culture medium in an amount selected from the group consisting of 0.1. Mu.M sdRNA/10,000 sdRNA/100. Mu.L medium, 0.5. Mu.M sdRNA/10,000 sdRNA/100. Mu.L medium, 0.75. Mu.M sdRNA/10,000 sdRNA/100. Mu.L medium, 1. Mu.M sdRNA/10,000 sdRNA/100. Mu.L medium, 1.25. Mu.M sdRNA/10,000 sdRNA/100. Mu.L medium, 1.5. Mu.M sdRNA/10,000 sdRNA/100. Mu.L medium, 2. Mu.M sdRNA/10,000 sdRNA/100. Mu.L medium, 5. Mu.M sdRNA/10,000 TIL/100. Mu.L medium, or 10. Mu.M sdRNA/10,000 TIL/100. Mu.L medium. In one embodiment, one or more sdrnas targeted to genes described herein, including PD-1, LAG-3, TIM-3, CISH, CBL-B, CD, HPK1, YAP1, and/or PTPN22, 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, prior to REP or during REP phases.

The oligonucleotide compositions of the invention, including the sdrnas, may be contacted with the TILs described herein during the amplification process, e.g., by dissolving the sdrnas in cell culture medium at high concentrations 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 dextran, electroporation, or by, for example, transfection using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art, such as those described in: U.S. patent No. 4,897,355; no. 5,459,127; 5,631,237; 5,955,365; 5,976,567; 10,087,464; 10,155,945; and Bergan et al, nucleic acids res.1993,21,3567, the disclosures of each of which are incorporated herein by reference in their entirety.

In some embodiments, more than one siRNA or sdRNA is used to reduce expression of a target gene. In some embodiments, more than one siRNA or sdRNA targeting PD-1, TIM-3, CBL-B, LAG-3, CISH, CD38, HPK1, YAP1, and/or PTPN22 is used together. In some embodiments, PD-1 siRNA or sdRNA is used with more than one of TIM-3, CBL-B, LAG-3 and/or CISH to reduce expression of more than one gene target. In some embodiments, a combination of LAG-3siRNA or sdRNA and CISH-targeted siRNA or sdRNA is used to reduce gene expression of both targets. In some embodiments, siRNAs or sdRNAs targeting more than one of PD-1, TIM-3, CBL-B, LAG-3, and/or CISH herein are available from Advirna LLC (WORS, mass.) or a number of other vendors.

In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), CD38, HPK1, YAP1, PTPN22, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), CD38, HPK1, YAP1, PTPN22, 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 LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), CD38, HPK1, YAP1, PTPN22, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from the group consisting of PD-1, LAG-3, CISH, CBL-B, TIM-3, and combinations thereof. In some embodiments, the siRNA or sdRNA targets a gene selected from one of PD-1 and LAG-3, CISH, CBL-B, TIM-3, and combinations thereof. In some embodiments, one siRNA or sdRNA targets PD-1 and one siRNA or sdRNA targets LAG-3. 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 CBL-B. In some embodiments, one siRNA or sdRNA targets LAG-3 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets LAG-3 and one siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets CISH and one siRNA or sdRNA targets CBL-B. In some embodiments, one siRNA or sdRNA targets TIM-3 and one siRNA or sdRNA targets PD-1. In some embodiments, one siRNA or sdRNA targets TIM-3 and one siRNA or sdRNA targets LAG-3. In some embodiments, one siRNA or sdRNA targets TIM-3 and one siRNA or sdRNA targets CISH. In some embodiments, one siRNA or sdRNA targets TIM-3 and one siRNA or sdRNA targets CBL-B.

As discussed herein, embodiments of the present invention provide genetically modified Tumor Infiltrating Lymphocytes (TILs) that are genetically edited to enhance therapeutic effects. Embodiments of the invention include gene editing by nucleotide insertion (RNA or DNA) into the TIL population to promote expression of more than one protein, to inhibit expression of more than one protein, and combinations thereof. Embodiments of the invention also provide methods for amplifying TIL into a therapeutic population, wherein the methods comprise gene editing of the TIL. There are several gene editing techniques that can be used to genetically modify the TIL population, which are suitable for use in the present invention. The methods include the methods described below and the viral and transposon methods described elsewhere herein. In one embodiment, the method of genetically modifying a TIL, MILs, or PBL to express CCR may also include inhibiting expression of the gene by stably knocking out the gene or temporarily knocking down the modification of the gene.

In one embodiment, the method comprises a method of genetically modifying a population of TILs in a first population, a second population, and/or a third population as described herein. In one embodiment, a method of genetically modifying a population of TILs includes the step of stably incorporating genes to produce or inhibit (e.g., silence) more than one protein. In one embodiment, the method of genetically modifying a population of TILs comprises an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, biophys.j.1991,60,297-306 and U.S. patent application publication No. 2014/0227237 A1, the respective disclosures of which are incorporated herein by reference in their entirety. Other electroporation methods known in the art may be used, such as described in 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 their entirety. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, 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 a defined and controlled permanent or temporary change in the TIL, comprising the step of applying at least three single, operator controlled, independently programmed sequences of DC electrical pulses to the TIL having a field strength equal to or greater than 100V/cm, wherein the at least three sequences of DC electrical pulses have 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) the second first pulse interval in the first set of at least three pulses is different from the second pulse interval in the second set of at least three pulses. In one embodiment, 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 at least three single, operator controlled, independently programmed sequences of DC electrical pulses to the TIL having a field strength equal to or greater than 100V/cm, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In one embodiment, 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 at least three single, operator controlled, independently programmed sequences of DC electrical pulses to the TIL having a field strength equal to or greater than 100V/cm, wherein at least two of the at least three pulses differ from each other in pulse width. In one embodiment, 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 a defined and controlled permanent or temporary change in the TIL, comprising the step of applying at least three single, operator controlled, independently programmed sequences of DC electrical pulses having a field strength equal to or greater than 100V/cm to the TIL, wherein a first pulse interval of a second of the first set of at least three pulses is different from a second pulse interval of a second of the second set of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method comprising the step of treating a TIL with a pulsed electric field to induce void formation in the TIL, comprising the step of applying a sequence of at least three DC electrical pulses having a field strength equal to or greater than 100V/cm to the TIL, wherein the sequence of at least three DC electrical 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) the first pulse interval of the second of the first set of at least three pulses is different from the second pulse interval of the second set of at least three pulses such that the induced holes last for a relatively long period of time and such that the viability of the TIL is maintained. In one embodiment, the method of genetically modifying a population of TILs comprises a calcium phosphate transfection step. The calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in 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, 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 their entirety. In one embodiment, the method of genetically modifying a population of TILs comprises a liposome transfection step. Liposome transfection methods such as 1 using the cationic lipids N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleylphospholipid ethanolamine (DOPE) in filtered water: methods of 1 (w/w) liposome formulation are known in the art and are described in Rose et al, biotechniques 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA,1987,84,7413-7417 and U.S. Pat. No. 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 their entirety. In one embodiment, a method of genetically modifying a population of TILs comprises using us patent 5,766,902;6,025,337;6,410,517; a transfection step of the methods described in 6,475,994 and 7,189,705; the respective disclosures of which are incorporated herein by reference in their entirety. The TIL may be a first population, a second population, and/or a third population of TILs as described herein.

According to embodiments, the gene editing process may include the use of programmable nucleases to mediate double-or single-strand break production at more than one immune checkpoint gene. Such programmable nucleases enable precise genome editing by introducing breaks at specific 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 targeting sequence. Double strand breaks in DNA subsequently attract endogenous repair mechanisms to the break sites to mediate genome editing through non-homologous end joining (NHEJ) or homology-directed repair (HDR). Thus, repair of a break can result in the introduction of an insertion/removal mutation that disrupts (e.g., quiesces, represses, or enhances) the target gene product.

Major nuclease types developed to enable site-directed 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 divided into two broad categories 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, e.g., cox et al, nature Medicine,2015, vol.21, no.2.

Non-limiting examples of gene editing methods that can be used in 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 for amplifying TIL into a therapeutic population may be performed according to any of the embodiments of the methods described herein (e.g., passage 2) or as described in U.S. patent application publication nos. US 2020/0299644 A1 and US 2020/011719 A1 and US patent No. 10,925,900 (the disclosures of which are incorporated herein by reference in their entirety), 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 one embodiment, the improved therapeutic effects of genetically edited TILs may be assessed by comparing them to unmodified TILs in vitro, for example by assessing in vitro effector functions, cytokine profiles, etc. compared to unmodified TILs. In certain embodiments, the method comprises gene editing the TIL population using CRISPR, TALE, and/or ZFN methods.

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 the AgilePoulse System or ECM 830 available from BTX-Harvard Apparatus, cellaxess Elektra (Cellectricon), nucleofector (Lonza/Amaxa), genePulser MXcell (BIORAD), iPrimar-96 (Primax), or SiPORTER96 (Ambion). In some embodiments of the invention, the electroporation system forms a closed sterile system with the remainder of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, forming a closed sterile system with the remainder of the TIL amplification 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., passage 2) or as described in U.S. patent application publication nos. US 2020/0299644 A1 and US 2020/011719 A1 and U.S. patent No. 10,925,900 (the disclosures of which are incorporated herein by reference in their entirety), wherein the method further comprises gene editing at least a portion of the TIL by a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf 1). According to a specific embodiment, the use of a CRISPR method during a TIL amplification procedure 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 aggregate regularly spaced 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 in the present invention: i, II and form III. Type II CRISPR (exemplified by Cas 9) is one of the most widely characterized systems.

CRISPR technology is a natural defense mechanism adapted 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 destroying the DNA of foreign invaders. CRISPR is a DNA-specific region with two unique features: there are nucleotide repeats and spacers present. The repeated sequences of nucleotides are distributed throughout the CRISPR region interspersed with short segments of foreign DNA (spacers) between the repeated sequences. In the type II CRISPR/Cas system, the spacer is integrated within the CRISPR locus and transcribed and processed to short CRISPR RNA (crRNA). These crrnas bind 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 pre-spacer adjacent motif (PAM) sequence containing dinucleotides upstream of the crRNA binding region. Whereby the CRISPR/Cas system can be re-targeted by redesigning the crRNA, and virtually any DNA sequence can be cleaved. The crRNA and tracrRNA in natural systems can be reduced to a single guide RNA (sgRNA) of about 100 nucleotides for genetic engineering. The CRISPR/Cas system can be moved directly to human cells by co-delivering a plasmid expressing Cas9 endonuclease and the necessary crRNA components. Different variants of Cas protein may be used to reduce targeting limitations (e.g., cas9 homologous genes, such as Cpf 1).

Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing TIL by CRISPR methods include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), cish, TGF beta, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP, 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, CY1B2, GU 1, GUX 3, SOX 1, KR 1, and KR 1, and PTOR 1.

Non-limiting examples of genes that can be enhanced by permanently gene editing TIL in CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15 and IL-21, pgc1α, NT-pgc1α, CXCR1, CXCR4, CXCR5, CXCR6, CCR1, CCR6, CCR7, CCR8, CCR9, CCR10, BATF and c-Jun.

Examples of systems, methods, and compositions for altering target gene sequence expression by CRISPR methods and useful in embodiments of the present invention are described in U.S. Pat. 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 in their entirety. Resources for performing CRISPR methods, such as plasmids for expressing CRISPR/Cas9 and CRISPR/Cpf1, are available from companies such as GenScript.

In one embodiment, genetic modification of a TIL population as described herein may be performed using the CRISPR/Cpf1 system as described in U.S. patent No. 9790490, the disclosure of which is incorporated herein by reference in its entirety.

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., passage 2) or as described in U.S. patent application publication nos. US 2020/0299644 A1 and US 2020/011719 A1 and U.S. patent No. 10,925,900 (the disclosures of which are incorporated herein by reference in their entirety), wherein the method further comprises gene editing at least a portion of the TIL by the TALE method. According to a specific embodiment, the use of the TALE method during the TIL amplification procedure 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 may also be referred to herein as the TALE method. TALE is a naturally occurring protein from the plant pathogenic bacterium, the genus Xanthomonas (Xanthomonas), containing a DNA binding domain consisting of a series of 33 to 35 amino acid repeat domains, each recognizing a single base pair. TALE specificity is determined by two hypervariable amino acids called repeated variable double Residues (RVD). Modular TALEs are repeatedly ligated together to identify contiguous DNA sequences. 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 make a targetable TALE nuclease. To induce site-directed mutagenesis, two respective TALEN arms separated by a 14 to 20 base pair spacer gene region pull fokl monomers closer to dimerize and create a target double strand break.

Several large, systematic studies using various assembly methods have indicated that TALE repeats can be combined to identify virtually any user-defined sequence. Custom designed TALE arrays are also commercially available from Cellectis Bioresearch (paris, france), transposagen Biopharmaceuticals (lekurd ston, 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 No. US 2011/0201118 A1; US 2013/017769 A1; US 2013/0315884 A1; US 2015/0203871 A1 and US 2016/012596 A1, the disclosures of each of which are incorporated herein by reference in their entirety.

Non-limiting examples of genes that can be silenced or repressed by permanently gene editing TIL in the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), cish, TGF beta, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP, 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, CY1B2, GU 1, GUX 3, SOX 1, KR 1, and KR 1, and PTOR 1.

Non-limiting examples of genes that can be enhanced by permanently gene editing TIL in TALE methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-21, pgc1α, NT-pgc1α, CXCR1, CXCR4, CXCR5, CXCR6, CCR1, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, and c-Jun.

Examples of systems, methods, and compositions for altering expression of a target gene sequence by the TALE method and useful in embodiments of the invention are described in U.S. patent No. 8,586,526, which is incorporated herein by reference in its entirety.

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. US 2020/0299644 A1 and US 2020/011719 A1 and US patent No. 10,925,900 (the disclosures of which are incorporated herein by reference in their entirety), wherein the method further comprises gene editing at least a portion of the TIL by zinc finger or zinc finger nuclease methods. According to particular embodiments, the use of zinc finger methods during the TIL amplification process results in quiescence or reduced 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 in the conserved ββα configuration contain about 30 amino acids. Several amino acids on the alpha helix surface typically contact 3bp in the DNA main groove and have different levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA binding domain, comprising eukaryotic transcription factors and contains zinc fingers. The second domain is a nuclease domain, comprising a fokl restriction enzyme and responsible for catalytic cleavage of DNA.

The DNA binding domain of each respective ZFN typically contains between three and six respective 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 that recognize 18 base pairs in total 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 a 3 base pair DNA sequence, to produce a 3-finger array that recognizes 9 base pair target sites. Alternatively, a selection-based approach such as Oligomeric Pool Engineering (OPEN) may be used to select a new zinc finger array from a random grouping library that accounts for context-dependent interactions between adjacent fingers. Engineered zinc fingers are available from Sangamo Biosciences (risman, california) and Sigma-Aldrich (st louis, miso, usa).

Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing TIL with zinc fingers include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), cish, TGF beta, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP, 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, CY1B2, GU 1, GUX 3, SOX 1, KR 1, and KR 1, KR 1 and PTOR 1.

Non-limiting examples of genes that can be enhanced by permanently gene editing TIL in a zinc finger approach include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, IL-21, pgc1α, NT-pgc1α, CXCR1, CXCR4, CXCR5, CXCR6, CCR1, CCR6, CCR7, CCR8, CCR9, CCR10, BATF, and c-Jun.

Examples of systems, methods, and compositions for altering expression of target gene sequences by zinc finger methods and useful in embodiments of the 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 in its entirety.

Other examples of systems, methods, and compositions for altering expression of a target gene sequence by zinc finger methods and useful in embodiments of the invention are described in beans et al, mol. Therapy,2015,23,1380-1390, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the TIL is optionally genetically engineered to include additional functionality, including but not limited to, high affinity TCRs, such as TCRs targeting tumor-associated antigens (e.g., MAGE-1, HER2, or NY-ESO-1), or Chimeric Antigen Receptors (CARs) that bind tumor-associated cell surface molecules (e.g., mesothelin) or cell line restricted cell surface molecules (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 cell line-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.

In some embodiments, the TILs of the present invention include TILs modified to express CCR and/or chemokine receptors, optionally genetically engineered to express membrane-bound IL-2, IL-12, IL-15, and/or IL-21, such as described in U.S. patent application publication No. US 2021/0052647 A1 or U.S. 2020/0172879 A1, the disclosures of which are incorporated herein by reference in their entirety.

E. Closed system for TIL manufacture

The present invention provides for the use of a closed system during the TIL cultivation process. The closed system allows prevention and/or reduction of microbial contamination, allows fewer flasks to be used and allows cost reduction. In some embodiments, the closure system uses two containers.

Closed systems are well known in the art and see, for example, http: htm and https: the// www.fda.gov/biologics Blood vaccines/guidelines company regulatoryinformation/guidelines/Blood/ucm 076779.Htm.

A sterile connection device (STCD) creates a sterile bond between two compatible pieces of tubing. This procedure allows sterile connection of a variety of containers and tube diameters. In some embodiments, the closure system includes a luer lock and heat seal system such as described in example G. In some embodiments, the containment system is accessed through a syringe under sterile conditions to maintain the sterility and containment characteristics of the system. In some embodiments, a closed system as described in example G is employed. In some embodiments, the TIL is formulated into a final product formulation container according to the methods described herein.

In some embodiments, the closed system uses only one container from the time tumor fragments are obtained until the TIL is ready for administration or cryopreservation to the patient. In some embodiments using two containers, 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 using two containers, the infusion bag is a HypoThermosol-containing infusion bag. The closed system or closed TIL cell culture system is characterized in that once the tumor sample and/or tumor fragments are added, the system can be tightly sealed from the outside to form a closed environment, which is not contaminated by 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 is reduced by 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 a significant reduction in microbial contamination.

In addition, the pH, partial pressure of carbon dioxide, and partial pressure of oxygen of the TIL cell culture environment each vary from cell culture to cell culture. Thus, even if the culture medium suitable for cell culture is circulated, the closed environment needs to be continuously maintained as an optimal environment for TIL proliferation. For this purpose, it is required to monitor physical factors of pH, partial pressure of carbon dioxide and partial pressure of oxygen in the culture liquid 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 the partial pressure of gas of the closed environment is instantaneously adjusted according to the change in the culture liquid to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system comprising a gas exchanger at the inlet to the closed system equipped with a monitoring device that measures the pH, partial pressure of carbon dioxide and partial pressure of oxygen of the closed environment, optimizing the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring device.

In some embodiments, the pressure in the enclosed environment is controlled continuously or intermittently. In other words, the pressure in the closed environment may be varied by, for example, a pressure maintenance device, thus ensuring that the space is suitable for TIL to grow in a positive pressure state, or promoting fluid to exude in a negative pressure state and thus promoting cell proliferation. Furthermore, by intermittently applying the negative pressure, it is possible to uniformly and effectively displace the liquid circulating in the closed environment by temporarily contracting the volume of the closed environment.

In some embodiments, optimal culture components for TIL proliferation may be substituted or added, and factors including, for example, IL-2 and/or OKT3, as well as combinations, may be added.

F. Optional TIL cryopreservation

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 that is collected after the second amplification. In some embodiments, cryopreservation occurs at the TIL in exemplary step F of fig. 1 and/or 8 (specifically, e.g., fig. 8B and/or 8C). In some embodiments, the TIL is stored frozen in an infusion bag. In some embodiments, the TIL is cryopreserved and then placed into an infusion bag. In some embodiments, the TIL is cryopreserved and not placed into an infusion bag. In some embodiments, the 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 into a chilled solution (e.g., 85% complement-deactivating AB serum and 15% dimethyl sulfoxide (DMSO)). The cell solution was placed in a frozen vial and stored at-80 ℃ for 24 hours, optionally transferred to a gaseous nitrogen freezer for cold storage. See Sadeghi et al, acta Oncologica 2013,52,978-986.

Where appropriate, the 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 substantially resuspended in complete medium and optionally washed more than once. In some embodiments, thawed TILs may be counted and viability assessed in a manner known in the art.

In a preferred embodiment, the TIL population is cryopreserved using CS10 cryopreservation media (CryoStor 10,BioLife Solutions). In a preferred embodiment, the TIL population is cryopreserved using a cryopreservation medium comprising Dimethylsulfoxide (DMSO). In a preferred embodiment, the TIL population uses 1:1 (volume: volume) ratio of CS10 and cell culture medium. In a preferred embodiment, the TIL population uses about 1:1 (volume: volume) ratio of CS10 and cell culture medium, and further comprising additional IL-2.

As discussed above and illustrated in steps a through E as provided in fig. 1 and/or 8 (specifically, e.g., fig. 8B and/or 8C), cryopreservation may occur at a number of points in the TIL amplification process. In some embodiments, the amplified TIL population after a first amplification, e.g., as provided according to step B, or after more than one second amplification, e.g., as per step D of fig. 1 or 8 (particularly, e.g., fig. 8B and/or fig. 8C), can be cryopreserved. Cryopreservation can typically be accomplished by placing the TIL population into a freezing solution (e.g., 85% complement-deactivating AB serum and 15% Dimethylsulfoxide (DMSO)). The cell solution was placed in a frozen vial and stored at-80 ℃ for 24 hours, optionally transferred to a gaseous nitrogen freezer for cold storage. See Sadeghi et al, 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 certain instances, the TIL population of step B may be immediately cryopreserved using the procedure discussed below. Alternatively, the subject TIL population may be subjected to step C and step D and then cryopreserved after step D. Similarly, in situations where a genetically modified TIL would be used in therapy, the population of TILs of step B or step D may be genetically modified for appropriate treatment.

G. Phenotypic characterization of amplified TIL

In some embodiments, TIL is analyzed for expression of a number of phenotypic markers after amplification, including those described herein and in the examples. In one embodiment, the expression of more than one phenotypic marker is examined. In some embodiments, the phenotypic characteristic of the TIL is analyzed after the first amplification of step B. In some embodiments, the phenotypic characteristics of the TIL are analyzed during the transition of step C. In some embodiments, the phenotypic characteristics of the TIL are analyzed during the transition according to step C and after cryopreservation. In some embodiments, the phenotypic characteristics of the TIL are analyzed after the second amplification according to step D. In some embodiments, the phenotypic characteristics of the TIL are analyzed after secondary or more than secondary amplification according to step D.

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, expression of CD8 and/or CD28 is high at TIL produced in accordance with the process of the invention as compared to other processes (e.g., as compared to 2A processes provided in, for example, fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C) at generation 3 processes provided in, for example, fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C)). In some embodiments, expression of CD8 is high at TIL produced in accordance with the process of the present invention as compared to other processes (e.g., as compared to 2A processes provided, for example, in fig. 8 (particularly, e.g., fig. 8B) at generation 3 processes provided, for example, in fig. 8 (particularly, e.g., fig. 8B and/or fig. 8C)). In some embodiments, expression of CD28 is high at TIL produced by a process according to the invention as compared to other processes (e.g., as compared to 2A processes provided, for example, in fig. 8 (particularly, e.g., fig. 8B and/or fig. 8C) at the 3 rd generation process provided, for example, in fig. 8 (particularly, e.g., fig. 8A)). In some embodiments, high CD28 expression is indicative of a younger, more durable TIL phenotype. In one embodiment, the expression of more than one regulatory marker is measured.

In one embodiment, during any step in the methods described herein for amplifying tumor-infiltrating lymphocytes (TILs), selecting the first TIL population, the second TIL population, the third TIL population, or the collected TIL population based on CD8 and/or CD28 expression is not performed.

In some embodiments, the percentage of central memory cells is high in TIL produced in accordance with the process of the present invention compared to other processes (e.g., in the 3 rd generation process provided, for example, in fig. 8 (particularly, e.g., fig. 8B and/or fig. 8C) compared to the 2A process provided, for example, in fig. 8 (particularly, e.g., fig. 8A)). In some embodiments, the memory signature of the central memory cell is selected from CCR7 and CD62L.

In some embodiments, the cd4+ and/or cd8+ TIL memory subsets may be separated 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 central memory (CM; CD45 RA-CD62L+) TILs. In some embodiments, the CD4+ and/or CD8+ TIL comprises an effector memory (EM; CD45RA-CD 62L-) TIL. In some embodiments, the CD4+ and/or CD8+ TILs comprise RA+ effector memory/effector cells (TEMRA/TEFF; CD45RA+CD62L+) TILs.

In some embodiments, the TIL expresses one or more markers selected from the group consisting of granulysin B, perforin, and granulysin. In some embodiments, TIL expresses granule lytic enzyme B. In some embodiments, the TIL expresses perforin. In some embodiments, TIL expresses granulysin.

In one embodiment, cytokine release of 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 by ELISA assay after a rapid second amplification step, after step D, e.g., provided in fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C). In some embodiments, TIL health is measured by IFN-gamma secretion. In some embodiments, IFN-gamma secretion is indicative of active TIL. In some embodiments, the potency assay for IFN-gamma production is used. IFN-gamma production is another measure of cytotoxic potential. IFN-gamma production can be measured by measuring the level of the cytokine IFN-gamma in TIL medium stimulated with antibodies against CD3, CD28 and CD137/4-1 BB. IFN-gamma levels in media from these stimulated TILs can be determined using measurement of IFN-gamma release. In some embodiments, an increase in the production of IFN- γ, e.g., at step D of the 3 rd generation process provided in fig. 8 (specifically, e.g., fig. 8B and/or fig. 8C), as compared to step D of the 2A process provided in fig. 8 (specifically, e.g., fig. 8A), indicates an increase in the cytotoxic potential of step D TIL. In some embodiments, IFN-gamma secretion is increased 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more than 5-fold. 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-fold. 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 of ex vivo TIL is measured. In some embodiments, IFN- γ is measured for ex vivo TIL, including TIL produced by the methods of the invention (including, e.g., the method of FIG. 8B).

In some embodiments, TIL capable of secreting at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold, or more than 5-fold IFN- γ is TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8B and/or fig. 8C). In some embodiments, a TIL capable of secreting at least 1-fold more IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8B and/or fig. 8C). In some embodiments, a TIL capable of secreting at least 2-fold more IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8B and/or fig. 8C). In some embodiments, the TIL capable of secreting at least 3-fold more IFN- γ is a TIL produced by the amplification methods of the invention (including, e.g., the methods of fig. 8B and/or fig. 8C). In some embodiments, a TIL capable of secreting at least 4-fold more IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8B and/or fig. 8C). In some embodiments, a TIL capable of secreting at least 5-fold more IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the methods of fig. 8B and/or fig. 8C).

The diverse 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 methods of producing TILs that exhibit and increase T cell reservoir diversity. In some embodiments, the TIL obtained by the present methods exhibits increased T cell reservoir diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell reservoir diversity compared to TILs prepared freshly collected and/or using methods other than those provided herein, including, for example, methods other than those embodied in fig. 8 (especially, e.g., fig. 8B and/or fig. 8C). In some embodiments, the TIL obtained by the present methods exhibits increased T cell reservoir diversity compared to freshly collected TIL and/or TIL prepared using a method referred to as generation 2 (as exemplified in fig. 8 (particularly, e.g., fig. 8A)). In some embodiments, the TIL obtained at 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 in the immunoglobulin, in the heavy chain of the immunoglobulin. In some embodiments, the diversity is in the immunoglobulin, in the immunoglobulin light chain. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is 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, the expression of TCRab (i.e., tcra/β) is increased. In some embodiments, the process described herein (e.g., the 3 rd generation process) exhibits higher clonal diversity based on the number of unique peptide CDRs in a sample (see, e.g., fig. 12-14) as compared to other processes (e.g., the process known as the 2 nd generation).

In some embodiments, activation and depletion of TIL may be determined by examining more than one marker. In some embodiments, the activation and depletion of TIL can be determined using polychromatic flow cytometry. In some embodiments, the activation and depletion markers include, but are not limited to, one or more markers 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, the activation and depletion markers include, but are not limited to, one or more markers selected from BTLA, CTLA-4, ICOS, ki67, LAG-3, PD-1, TIGIT and/or TIM-3. In some embodiments, the activation and depletion markers include, but are not limited to, one or more markers selected from 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 assayed and/or analyzed to examine T cell activation, inhibition, or function. In some embodiments, the T cell markers may include, but are not limited to, one or more markers selected from TIGIT, CD3, foxP3, tim-3, PD-1, CD103, CTLA-4, LAG-3, BTLA-4, ICOS, ki67, CD8, CD25, CD45, CD4, and/or CD 59.

In some embodiments, the phenotypic identification is checked after cryopreservation.

H. Additional process embodiment

In some embodiments, the invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) Obtaining a first population of TILs from a resected tumor of a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Performing an initial first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein the initial 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) Generating a third population of TILs by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs) for a rapid second expansion, 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; and (d) collecting the therapeutic TIL population obtained from step (c). In some embodiments, the step of rapid second amplification is divided into the following steps to achieve a longitudinal expansion of the culture scale: (1) Performing a rapid second amplification by culturing a second TIL population in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days or about 2 to 4 days, followed by (2) transferring 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 the second TIL population from the small scale culture is cultured in the larger scale culture in the second vessel for a period of about 4 to 7 days or about 4 to 8 days. In some embodiments, the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale: (1) Performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (2) transferring and partitioning 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 vessels of equal size to the first vessel, wherein in each second vessel, the portion of the second TIL population from the first small-scale culture transferred to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (1) Performing a rapid second amplification by culturing the second TIL population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days or about 2 to 4 days, followed by (2) transferring and partitioning 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 vessels (e.g., G-REX 500MCS vessels) that are larger in size than the first vessel, wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (1) Rapid second expansion is performed by culturing the second TIL population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (2) transferring and partitioning the second TIL population from the first small-scale culture into 2, 3, or 4 second vessels (e.g., G-REX 500MCS vessels) that are larger in size than the first vessel, wherein in each second vessel, the portion of the second TIL population transferred from the small-scale culture to the second vessel is cultured in the larger-scale culture for a period of about 5 to 7 days.

In some embodiments, the invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) Obtaining a first population of TILs from a resected tumor of a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Performing an initial first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein the initial first amplification is performed for 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) Generating a third population of TILs by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs) for a rapid second expansion, 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; and (d) collecting the therapeutic TIL population obtained from step (c). In some embodiments, the step of rapid second amplification is divided into the following steps to achieve a longitudinal expansion of the culture scale: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 2 to 4 days, followed by (2) transferring 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 the second TIL population from the small scale culture is cultured in the second vessel for a period of about 4 to 8 days in a larger scale culture. In some embodiments, the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale: (1) Performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 2 to 4 days, followed by (2) transferring and partitioning 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 vessels of equal size to the first vessel, wherein in each second vessel the portion of the second TIL population from the first small-scale culture transferred to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (1) Performing a rapid second amplification by culturing the second TIL population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 2 to 4 days, followed by (2) transferring and partitioning 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 vessels (e.g., G-REX 500MCS vessels) that are larger in size than the first vessel, wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (1) Rapid second expansion is performed by culturing the second TIL population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (2) transferring and partitioning the second TIL population from the first small-scale culture into 2, 3, or 4 second vessels (e.g., G-REX 500MCS vessels) that are larger in size than the first vessel, wherein in each second vessel, the portion of the second TIL population transferred from the small-scale culture to the second vessel is cultured in the larger-scale culture for a period of about 4 to 5 days.

In some embodiments, the invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) Obtaining a first population of TILs from a resected tumor of a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Performing an initial first amplification by culturing a first population of TILs in a cell culture medium comprising IL-2 and OKT-3, wherein the initial 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) Generating a third population of TILs by contacting the second population of TILs with a cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs) for a rapid second expansion, 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; and (d) collecting the therapeutic TIL population obtained from step (c). In some embodiments, the step of rapid second amplification is divided into the following steps to achieve a longitudinal expansion of the culture scale: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (2) transferring 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 the second TIL population from the small scale culture is cultured in the second vessel for a period of about 4 to 7 days in a larger scale culture. In some embodiments, the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale: (1) Performing a rapid second amplification by culturing a second TIL population in a first small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (2) transferring and partitioning 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 vessels of equal size to the first vessel, wherein in each second vessel, the portion of the second TIL population from the first small-scale culture transferred to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (1) Performing a rapid second amplification by culturing the second TIL population in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (2) transferring and partitioning 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 vessels (e.g., G-REX 500MCS vessels) that are larger in size than the first vessel, wherein in each second vessel the portion of the second TIL population transferred from the small scale culture to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (1) Rapid second expansion is performed by culturing the second TIL population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 4 days, followed by (2) transferring and partitioning the second TIL population from the first small-scale culture into 2, 3, or 4 second vessels (e.g., G-REX 500MCS vessels) that are larger in size than the first vessel, wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to the second vessel is cultured in the larger-scale culture for a period of about 5 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the primary first amplification is performed by contacting the first population of TILs with a medium further comprising exogenous Antigen Presenting Cells (APCs), the number of APCs of the medium of step (c) being greater than the number of APCs of the medium of step (b).

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to just or about 20: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to just or about 10: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 9: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to just or about 8: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to just or about 7: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 6: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 5: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to just or about 4: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to just or about 3: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.9: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.8: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.7: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.6: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.5: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.4: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.3: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.2: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2.1: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 1.1:1 to exactly or about 2: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to just or about 10: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 5: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to just or about 4: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to just or about 3: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.9: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.8: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.7: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.6: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.5: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.4: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.3: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.2: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 exactly or about 2:1 to exactly or about 2.1: 1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 another embodiment, the invention provides a modified method as described in any preceding paragraph 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 another embodiment, the invention provides a modified, as applicable, aboveThe method of any preceding paragraph, wherein the number of APCs added in the primary first amplification is exactly or about 1X 10 ⁸ 、1.1×10 ⁸ 、1.2×10 ⁸ 、1.3×10 ⁸ 、1.4×10 ⁸ 、1.5×10 ⁸ 、1.6×10 ⁸ 、1.7×10 ⁸ 、1.8×10 ⁸ 、1.9×10 ⁸ 、2×10 ⁸ 、2.1×10 ⁸ 、2.2×10 ⁸ 、2.3×10 ⁸ 、2.4×10 ⁸ 、2.5×10 ⁸ 、2.6×10 ⁸ 、2.7×10 ⁸ 、2.8×10 ⁸ 、2.9×10 ⁸ 、3×10 ⁸ 、3.1×10 ⁸ 、3.2×10 ⁸ 、3.3×10 ⁸ 、3.4×10 ⁸ Or 3.5X10 ⁸ The number of APCs added in the rapid second amplification is exactly or about 3.5X10 ⁸ 、3.6×10 ⁸ 、3.7×10 ⁸ 、3.8×10 ⁸ 、3.9×10 ⁸ 、4×10 ⁸ 、4.1×10 ⁸ 、4.2×10 ⁸ 、4.3×10 ⁸ 、4.4×10 ⁸ 、4.5×10 ⁸ 、4.6×10 ⁸ 、4.7×10 ⁸ 、4.8×10 ⁸ 、4.9×10 ⁸ 、5×10 ⁸ 、5.1×10 ⁸ 、5.2×10 ⁸ 、5.3×10 ⁸ 、5.4×10 ⁸ 、5.5×10 ⁸ 、5.6×10 ⁸ 、5.7×10 ⁸ 、5.8×10 ⁸ 、5.9×10 ⁸ 、6×10 ⁸ 、6.1×10 ⁸ 、6.2×10 ⁸ 、6.3×10 ⁸ 、6.4×10 ⁸ 、6.5×10 ⁸ 、6.6×10 ⁸ 、6.7×10 ⁸ 、6.8×10 ⁸ 、6.9×10 ⁸ 、7×10 ⁸ 、7.1×10 ⁸ 、7.2×10 ⁸ 、7.3×10 ⁸ 、7.4×10 ⁸ 、7.5×10 ⁸ 、7.6×10 ⁸ 、7.7×10 ⁸ 、7.8×10 ⁸ 、7.9×10 ⁸ 、8×10 ⁸ 、8.1×10 ⁸ 、8.2×10 ⁸ 、8.3×10 ⁸ 、8.4×10 ⁸ 、8.5×10 ⁸ 、8.6×10 ⁸ 、8.7×10 ⁸ 、8.8×10 ⁸ 、8.9×10 ⁸ 、9×10 ⁸ 、9.1×10 ⁸ 、9.2×10 ⁸ 、9.3×10 ⁸ 、9.4×10 ⁸ 、9.5×10 ⁸ 、9.6×10 ⁸ 、9.7×10 ⁸ 、9.8×10 ⁸ 、9.9×10 ⁸ Or 1X 10 ⁹ And (3) APC.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the number of APCs added in the primary first amplification is selected from exactly or about 1X 10 ⁸ To exactly or about 3.5X10 APC ⁸ The number of APCs added in the rapid second amplification is selected from the range of just or about 3.5X10 ⁸ APC to exactly or about 1X 10 ⁹ Ranges of APC.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the number of APCs added in the primary first amplification is selected from exactly or about 1.5X10 ⁸ To exactly or about 3X 10 APC ⁸ The number of APCs added in the rapid second amplification is selected from the range of just or about 4X 10 APCs ⁸ To exactly or about 7.5X10 APC ⁸ Ranges of APC.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the number of APCs added in the primary first amplification is selected from the group consisting of exactly or about 2X 10 ⁸ To exactly or about 2.5X10 APC ⁸ The number of APCs added in the rapid second amplification is selected from the range of just or about 4.5X10 ⁸ To exactly or about 5.5X10 APC ⁸ Ranges of APC.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein, exactly or about 2.5X10 ⁸ The APCs were added to the primary first amplification, just or about 5X 10 ⁸ The APCs were added to the rapid second amplification.

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable 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 TIL therapeutic populations from the plurality of containers of step (c) are combined to produce the collected population of TILs from step (d).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of tumors are equally distributed in a plurality of separate containers.

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of separate containers comprises two to twenty separate containers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of separate containers comprises from twenty to fifteen separate containers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of separate containers comprises two to ten separate containers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of separate containers comprises two to five separate containers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the initial first amplification is performed on a first population of TILs of step (b) in each container and the rapid second amplification of step (c) is performed on a second population of TILs generated from the first population of TILs in the same container.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the separate containers each comprise a first vapor permeable surface area.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of tumor fragments are distributed in a single container.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the single container comprises a first vapor permeable surface area.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), the number of APCs added in step (c) being greater than the number of APCs added in step (b), in step (b) APCs being layered on the first air permeable surface area at an average thickness of exactly or about one cell layer to exactly or about three cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b), the APC is layered on the first air permeable surface area with an average thickness of exactly or about 1.5 cell layers to exactly or about 2.5 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b), the APC is layered on the first air permeable surface area with an average thickness of exactly or about 2 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b), the APC is layered on the first air permeable surface area with an average thickness of exactly 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered on the first air permeable surface area with an average thickness of from exactly or about 3 cell layers to exactly or about 10 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered on the first air permeable surface area with an average thickness of from exactly or about 4 cell layers to exactly or about 8 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered on the first air permeable surface area with an average thickness of exactly or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered on the first air permeable surface area with an average thickness of exactly or about 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 another embodiment, 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 area, and in step (c) the rapid second amplification is performed in a second vessel comprising a second gas-permeable surface area.

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph where applicable, wherein in step (b) the APC is layered on the first air permeable surface area with an average thickness of exactly 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered on the second air permeable surface area with an average thickness of from exactly or about 3 cell layers to exactly or about 10 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered on the second air permeable surface area with an average thickness of from exactly or about 4 cell layers to exactly or about 8 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (c), the APC is layered on the second air permeable surface area with an average thickness of exactly or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph where applicable, wherein in step (c) the APC is layered on the second air permeable surface area with an average thickness of exactly or about 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 another embodiment, 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 area, and in step (c) the rapid second amplification is performed in the first vessel.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), the number of APCs added in step (c) being greater than the number of APCs added in step (b), in step (b) APCs being layered on the first air permeable surface area with an average thickness of from exactly or about 1 cell layer to exactly or about 3 cell layers.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 10.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 9.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 8.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 7.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 6.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 5.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 4.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 3.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.1 to just or about 1: 2.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.2 to just or about 1: 8.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.3 to just or about 1: 7.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.4 to just or about 1: 6.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.5 to just or about 1: 5.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.6 to just or about 1: 4.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.7 to just or about 1: 3.5.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.8 to just or about 1:3.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:1.9 to just or about 1: 2.5.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs in step (b) to the average number of stacked APCs in step (c) being selected from the group consisting of exactly or about 1:2.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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), 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 stacked APCs to the average number of stacked APCs in step (b) being selected from exactly 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 another embodiment, the invention provides a modified method as described in any preceding paragraph 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 1.5:1 to exactly or about 100:1.

in another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 50:1.

in another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 25:1.

in another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 20:1.

in another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is exactly or about 10:1.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the second TIL population is at least exactly or about 50 times higher in number than the first TIL population.

In another embodiment, the invention provides a modified method as described in any of the preceding paragraphs that apply above, wherein the second TIL population is at least exactly 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 another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the cell culture medium is supplemented with additional IL-2 for exactly or about 2 days or exactly or about 3 days after the start of the second time period in step (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, further comprising the step of cryopreserving the collected TIL population in step (d) using a cryopreservation process.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, to include the additional step (e) of transferring the collected population of TILs from step (d) to an infusion bag optionally containing HypoThermosol, after step (d).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, to include the step of cryopreserving the infusion bag containing the collected TIL population in step (e) using a cryopreservation process.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the method is used in the form of 1:1 ratio of the collected TIL population to the cryopreservation media for the cryopreservation process.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the PBMCs are irradiated and allogenic.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the total number of APCs added to the cell culture in step (b) is 2.5X10 ⁸ And each.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the total number of APCs added to the cell culture in step (c) is 5X 10 ⁸ And each.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the APC is PBMC.

In another embodiment, 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 another embodiment, 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 another embodiment, 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 LOVO cell processing system.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 5 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 10 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 15 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 20 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 25 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 30 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 35 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 40 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 45 to exactly or about 60 fragments per container in step (b).

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each segment has a length of exactly or about 27mm ³ Is a volume of (c).

In another embodiment, the invention provides a modifiedThe method of any preceding paragraph above where the fragments have a diameter of exactly or about 20mm ³ To exactly or about 50mm ³ Is a volume of (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each segment has a length of exactly or about 21mm ³ To just or about 30mm ³ Is a volume of (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each segment has a length of exactly or about 22mm ³ To exactly or about 29.5mm ³ Is a volume of (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each segment has a length of exactly or about 23mm ³ To exactly or about 29mm ³ Is a volume of (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each segment has a length of exactly or about 24mm ³ To exactly or about 28.5mm ³ Is a volume of (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each segment has a length of exactly or about 25mm ³ To just or about 28mm ³ Is a volume of (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each segment has a length of exactly or about 26.5mm ³ To just or about 27.5mm ³ Is a volume of (c).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein each fragment has exactly 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 Is a volume of (c).

In another embodiment, the invention provides a modified application as aboveThe method of any preceding paragraph, wherein the plurality of fragments comprises exactly or about 30 to exactly or about 60 fragments, the total volume being exactly or about 1300mm ³ To just or about 1500mm ³ 。

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the plurality of fragments comprises exactly or about 50 fragments, the total volume being exactly or about 1350mm ³ 。

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the cell culture medium is provided in a container which is a G-container or a Xuri cell bag.

In another embodiment, 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 another embodiment, 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 about 6,000IU/mL.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the cryopreservation medium comprises Dimethylsulfoxide (DMSO).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the cryopreservation medium comprises 7% to 10% DMSO.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the second time period in step (c) is performed over a time period 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 another embodiment, 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 separately over a period of time of exactly or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In another embodiment, 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 separately over a period of time of exactly or about 5 days, 6 days, or 7 days.

In another embodiment, 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 separately conducted over a period of time of exactly or about 7 days.

In another embodiment, 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 exactly or about 14 days to exactly or about 18 days.

In another embodiment, 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 exactly or about 15 days to exactly or about 18 days.

In another embodiment, 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 exactly or about 16 days to exactly or about 18 days.

In another embodiment, 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 exactly or about 17 days to exactly or about 18 days.

In another embodiment, 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 exactly or about 14 days to exactly or about 17 days.

In another embodiment, 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 exactly or about 15 days to exactly or about 17 days.

In another embodiment, 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 exactly or about 16 days to exactly or about 17 days.

In another embodiment, 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 exactly or about 14 days to exactly or about 16 days.

In another embodiment, 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 exactly or about 15 days to exactly or about 16 days.

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) to (d) are performed for exactly or about 15 days in total.

In another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) to (d) are performed for exactly or about 16 days in total.

In another embodiment, 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 exactly or about 17 days.

In another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein steps (a) to (d) are performed for exactly or about 18 days in total.

In another embodiment, 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 exactly or about 14 days or less than 14 days.

In another embodiment, 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 or less in total.

In another embodiment, 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 exactly or about 16 days or less than 16 days.

In another embodiment, 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 exactly or about 18 days or less than 18 days.

In another embodiment, 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 TILs sufficient for a therapeutically effective dose of TILs.

In another embodiment, the invention provides a modified method as described in any preceding paragraph applicable above, wherein the amount of TIL sufficient for a therapeutically effective dose is exactly or about 2.3 x 10 ¹⁰ Up to just or about 13.7X10 ¹⁰ And each.

In another embodiment, the invention provides a modified method as described in any preceding paragraph applicable above, wherein the third TIL population in step (c) provides increased efficacy, increased interferon gamma production, and/or increased polyclonality.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the third TIL population in step (c) provides at least 1-fold to 5-fold or more than 5-fold interferon-gamma production compared to TIL prepared by a process longer than 16 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the third TIL population in step (c) provides at least 1-fold to 5-fold or more than 5-fold interferon-gamma production compared to TIL prepared by a process longer than 17 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the third TIL population in step (c) provides at least 1-fold to 5-fold or more than 5-fold interferon-gamma production compared to TIL prepared by a process longer than 18 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable 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 cell population of step (b).

In another embodiment, the invention provides a modified process as described in any preceding paragraph as applicable above, wherein each container recited in the process is a closed container.

In another embodiment, 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 another embodiment, 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 another embodiment, 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 another embodiment, 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 another embodiment, the invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) made by the method described in any preceding paragraph applicable as above.

In another embodiment, 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) or OKT 3.

In another embodiment, 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 relative to TILs prepared by a process in which first expansion of TILs is performed in the absence of any added Antigen Presenting Cells (APCs).

In another embodiment, 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 relative to TILs prepared by a process in which first amplification of TILs is performed in the absence of any added OKT 3.

In another embodiment, 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 wherein first expansion of TILs is performed in the absence of added Antigen Presenting Cells (APCs) and in the absence of added OKT 3.

In another embodiment, 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 longer than 16 days.

In another embodiment, 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 longer than 17 days.

In another embodiment, 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 longer than 18 days.

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs as applicable above, the therapeutic TIL population providing increased production of interferon gamma.

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs as applicable above, the therapeutic TIL population providing increased polyclonality.

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs as applicable above, the therapeutic TIL population providing increased efficacy.

In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 1-fold more interferon-gamma than TILs prepared by a process longer than 16 days. In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 1-fold more interferon-gamma than TILs prepared by a process longer than 17 days. In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 1-fold more interferon-gamma than TILs prepared by a process longer than 18 days. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 1-fold more interferon-gamma.

In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 2-fold more interferon-gamma than TILs prepared by a process longer than 16 days. In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 2-fold more interferon-gamma than TILs prepared by a process longer than 17 days. In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 2-fold more interferon-gamma than TILs prepared by a process longer than 18 days. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 2-fold more interferon-gamma.

In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 3-fold more interferon-gamma than TILs prepared by a process longer than 16 days. In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 3-fold more interferon-gamma than TILs prepared by a process longer than 17 days. In another embodiment, the invention provides a modified population of therapeutic TILs as described in any of the preceding paragraphs as applicable above, the population of therapeutic TILs being capable of producing at least 3-fold more interferon-gamma than TILs prepared by a process longer than 18 days. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 3-fold more interferon-gamma.

In another embodiment, the invention provides a population of therapeutic Tumor Infiltrating Lymphocytes (TILs) capable of producing at least 1-fold more interferon-gamma than TILs prepared by a process in which the first expansion of TILs is performed in the absence of any added Antigen Presenting Cells (APCs). In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 1-fold more interferon-gamma.

In another embodiment, the invention provides a population of therapeutic Tumor Infiltrating Lymphocytes (TILs) capable of producing at least 1-fold more interferon-gamma than TILs prepared by a process in which the first amplification of TILs is performed in the absence of any added OKT 3. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 1-fold more interferon-gamma.

In another embodiment, the invention provides a therapeutic TIL population capable of producing at least 2-fold more interferon-gamma than TIL prepared by a process in which the first amplification of TIL is performed in the absence of any added APC. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 2-fold more interferon-gamma.

In another embodiment, the invention provides a therapeutic TIL population capable of producing at least 2-fold more interferon-gamma than TIL prepared by a process in which the first amplification of TIL is performed in the absence of any added OKT 3. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 2-fold more interferon-gamma.

In another embodiment, the invention provides a therapeutic TIL population capable of producing at least 3-fold more interferon-gamma than TIL prepared by a process in which the first amplification of TIL is performed in the absence of any added APC. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 1-fold more interferon-gamma.

In another embodiment, the invention provides a therapeutic TIL population capable of producing at least 3-fold more interferon-gamma than TIL prepared by a process in which the first amplification of TIL is performed in the absence of any added OKT 3. In some embodiments, because the amplification process described herein, e.g., as described in or according to steps a-F above (as also shown, e.g., in fig. 8 (and in particular, e.g., fig. 8B and/or fig. 8C)), TIL is capable of producing at least 3-fold more interferon-gamma.

In another embodiment, 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 core biopsy, a core needle biopsy (core needle biopsy), or a fine needle aspirate.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the tumor fragment is a core biopsy.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the tumor fragments are fine needle aspirates.

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the tumor fragment is a core needle biopsy.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein (i) the method comprises obtaining one or more small biopsy (including, for example, a perforated biopsy), a core biopsy, a core needle biopsy, or a first population of TILs of fine needle aspirates from tumor tissue of a subject; (ii) The method comprises performing the step of culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days prior to performing the step of initial first amplification; (iii) The method comprises performing an initial 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein (i) the method comprises obtaining one or more small biopsy (including, for example, a perforated biopsy), a core biopsy, a core needle biopsy, or a first population of TILs of fine needle aspirates from tumor tissue of a subject; (ii) The method comprises performing the step of culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days prior to performing the step of initial first amplification; (iii) The method comprises performing an initial first amplification for a period of about 8 days; and (iv) the method comprises performing a rapid second amplification by culturing the second TIL population for about 5 days, sub-culturing the culture in split flasks (split) for up to 5 sub-cultures, and culturing the sub-cultures for about 6 days. In some of the foregoing embodiments, at most 5 subcultures are each cultured in a vessel of the same size or larger than the vessel in which the second TIL population began to culture in the rapid second amplification. In some of the foregoing embodiments, the culturing of the second TIL population is aliquoted into up to 5 subcultures. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 20 small biopsy sections (including, for example, perforated biopsy sections), core biopsy sections, core needle biopsy sections, or fine needle aspirates of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 10 small biopsy sections (including, for example, perforated biopsy sections), core biopsy sections, core needle biopsy sections, or fine needle aspirates of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph applicable above, wherein 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 core biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein 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 core biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 20 core biopsy sections of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 10 core biopsy sections of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein 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 biopsy of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 core biopsy sections of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 20 fine needle aspirates of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 10 fine needle aspirates of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein 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 of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fine needle aspirates of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 20 core needle biopsy sections of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 10 core needle biopsy sections of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein 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.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 core needle biopsy sections of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 20 small biopsy sections (including, for example, perforated biopsy sections) of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first TIL population is obtained from 1 to about 10 small biopsy sections (including, for example, perforated biopsy sections) of tumor tissue of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph applicable above, wherein 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 of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein 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 of the subject.

In another embodiment, the invention provides a modified method as described in any preceding paragraph applicable above, wherein (i) the method comprises obtaining a first population of TIL of 1 to about 10 core biopsy sections from tumor tissue of the subject; (ii) The method comprises performing the step of culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days prior to performing the step of initial first amplification; (iii) The method comprises performing an initial first amplification step by culturing a first population of TILs in a 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; and (iv) the method comprises performing a rapid second amplification step by culturing the second TIL population in a 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 another embodiment, the invention provides a modified method as described in any preceding paragraph applicable above, wherein (i) the method comprises obtaining a first population of TIL of 1 to about 10 core biopsy sections from tumor tissue of the subject; (ii) The method comprises performing the step of culturing the first TIL population in a cell culture medium comprising IL-2 for a period of about 3 days prior to performing the step of initial first amplification; (iii) The method comprises performing an initial first amplification step by culturing a first population of TILs in a 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; and (iv) the method comprises performing rapid second amplification by culturing the second TIL population in a medium comprising IL-2, OKT-3 and APC for about 5 days, plating the culture medium into up to 5 subcultures, and culturing the subcultures in a medium comprising IL-2 for about 6 days each. In some of the foregoing embodiments, at most 5 subcultures are each cultured in a vessel of the same size or larger than the vessel in which the second TIL population began to culture in the rapid second amplification. In some of the foregoing embodiments, the culturing of the second TIL population is aliquoted into up to 5 subcultures. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph applicable above, wherein (i) the method comprises obtaining a first population of TIL of 1 to about 10 core biopsy sections from tumor tissue of the subject; (ii) The method comprises the step of culturing the first TIL population in a G-Rex 100M flask for a period of about 3 days in a cell culture medium comprising 6000IU IL-2/mL CM1 medium in 0.5L prior to the step of performing the initial first amplification; (iii) The method comprises adding a solution containing 6000IU/mL IL-2 and 30ng/mL OKT-3 and about 10 ⁸ Initial first expansion was performed by incubating 0.5L CM1 medium of feeder cells for a period of about 8 days; and (iv) the method comprises performing a rapid second amplification by: (a) Transferring the second TIL population to a medium containing 5L of CM2 and 3000IU/mL IL-2, 30ng/mL OKT-3 and 5X 10 ⁹ G-Rex500MCS flask of individual feeder cells and cultured for about 5 days, (b) by incubating 10 ⁹ Each TIL was transferred to each of up to 5G-Rex 500MCS flasks containing 5L of AIM-V medium and 3000IU/mL IL-2 to flask culture nutrients into up to 5 subcultures and the culture subcultures were performed for about 6 days. In some of the foregoing embodiments, the steps of the method are completed within about 22 days.

In another embodiment, the invention provides a method of expanding T cells comprising: (a) Performing an initial first expansion of a first T cell population by culturing the first T cell population obtained from the donor to grow the first T cell population 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 such that the first T cell population grows and the activation of the first T cell population is enhanced to obtain a second T cell population; and (c) collecting the second T cell population. In another embodiment, the step of rapid second amplification is divided into the following steps to achieve a longitudinal expansion of the culture scale: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (b) transferring the first T cell population from the small scale culture to a second vessel (e.g., G-REX 500MCS vessel) that is larger than the first vessel and culturing the first T cell population from the small scale culture in a larger scale culture in the second vessel for a period of about 4 to 7 days. In some embodiments, the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale: (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-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (b) transferring and partitioning 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 equal size to the first vessel, wherein in each second vessel the portion of the first T cell population from the first small-scale culture transferred to the second vessel is cultured in a second small-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapidly expanding is divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (b) transferring and partitioning 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 vessels (e.g., G-REX 500MCS vessels) of larger size than the first vessel, wherein in each second vessel the portion of the first T cell population from the small-scale culture transferred to the second vessel 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 divided into the following steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Rapid second expansion is performed by culturing the first T cell population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 4 days, followed by (b) transferring and partitioning the first T cell population 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, wherein in each second vessel the portion of the first T cell population from the small-scale culture transferred to the second vessel is cultured in the larger-scale culture for a period of about 5 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the step of rapidly second amplifying is divided into a plurality of steps to achieve a longitudinal expansion of the culture scale: (a) Performing a rapid second expansion by culturing the first T cell population in a small scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 2 to 4 days, followed by (b) transferring the first T cell population from the small scale culture to a second vessel (e.g., G-REX 500MCS vessel) that is larger than the first vessel and culturing the first T cell population from the small scale culture in a larger scale culture in the second vessel for a period of about 5 to 7 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale: (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-REX 100MCS vessel) for a period of about 2 to 4 days, followed by (b) transferring and partitioning 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 equal size to the first vessel, wherein in each second vessel the portion of the first T cell population from the first small-scale culture transferred to the second vessel is cultured in a second small-scale culture for a period of about 5 to 7 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 2 to 4 days, followed by (b) transferring and partitioning 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 vessels (e.g., G-REX 500MCS vessels) of a size greater than the first vessel, wherein in each second vessel the portion of the first T cell population from the small-scale culture transferred to the second vessel is cultured in a larger-scale culture for a period of about 5 to 7 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (b) transferring and partitioning the first T cell population from the small-scale culture into 2, 3 or 4 second vessels (e.g., G-REX 500MCS vessels) of a size larger than the first vessel, wherein in each second vessel the portion of the first T cell population from the small-scale culture transferred to the second vessel is cultured in a larger-scale culture for a period of about 5 to 6 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Rapid second expansion is performed by culturing the first T cell population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (b) transferring and dispensing the first T cell population from the small-scale culture into 2, 3, or 4 second vessels (e.g., G-REX 500MCS vessels) of a size larger than the first vessel, wherein in each second vessel the portion of the first T cell population from the small-scale culture transferred to the second vessel is cultured in a larger-scale culture for a period of about 5 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Rapid second expansion is performed by culturing the first T cell population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (b) transferring and dispensing the first T cell population from the small-scale culture into 2, 3, or 4 second vessels (e.g., G-REX 500MCS vessels) of a size larger than the first vessel, wherein in each second vessel the portion of the first T cell population from the small-scale culture transferred to the second vessel is cultured in a larger-scale culture for a period of about 6 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the step of rapidly expanding is divided into a plurality of steps to achieve lateral expansion of the culture scale and longitudinal expansion of the scale: (a) Rapid second expansion is performed by culturing the first T cell population in a small-scale culture in a first vessel (e.g., G-REX 100MCS vessel) for a period of about 3 to 4 days, followed by (b) transferring and dispensing the first T cell population from the small-scale culture into 2, 3, or 4 second vessels (e.g., G-REX 500MCS vessels) of a size larger than the first vessel, wherein in each second vessel the portion of the first T cell population from the small-scale culture transferred to the second vessel is cultured in a larger-scale culture for a period of about 7 days.

In another embodiment, 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 over a period of up to 7 days.

In another embodiment, 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 over a period of up to 8 days.

In another embodiment, 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 over a period of up to 9 days.

In another embodiment, 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 over a period of up to 10 days.

In another embodiment, 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 over a period of up to 11 days.

In another embodiment, 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 over a period of 7 days and the rapid second amplification of step (b) is performed over a period of up to 9 days.

In another embodiment, 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 over a period of 7 days and the rapid second amplification of step (b) is performed over a period of up to 10 days.

In another embodiment, 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 over a period of 7 days or 8 days and the rapid second amplification of step (b) is performed over a period of up to 9 days.

In another embodiment, 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 over a period of 7 days or 8 days and the rapid second amplification of step (b) is performed over a period of up to 10 days.

In another embodiment, 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 over a period of 8 days and the rapid second amplification of step (b) is performed over a period of up to 9 days.

In another embodiment, 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 over a period of 8 days and the rapid second amplification of step (b) is performed over a period of up to 8 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (a) the first population of T cells is cultured in a first medium comprising OKT-3 and IL-2.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first medium comprises a 4-1BB agonist, OKT-3 and IL-2.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first medium comprises OKT-3, IL-2 and Antigen Presenting Cells (APCs).

In another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the first medium comprises a 4-1BB agonist, OKT-3, IL-2 and Antigen Presenting Cells (APC).

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein in step (b) the first population of T cells is cultured in a second medium comprising OKT-3, IL-2 and Antigen Presenting Cells (APC).

In another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the second medium comprises a 4-1BB agonist, OKT-3, IL-2 and Antigen Presenting Cells (APC).

In another embodiment, 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 on 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 APC, the second population of APCs being layered on the first gas permeable surface, the second population of APCs being greater than the first population of APCs.

In another embodiment, the invention provides a modified method as described in any preceding paragraph that applies 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 donor of the first population of T cells, the first population of APCs being layered on 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 on the first gas permeable surface, the second population of APCs being greater than the first population of APCs.

In another embodiment, 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 on 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 the donor of the first population of APCs, the second population of APCs being layered on the first gas permeable surface, the second population of APCs being greater than the first population of APCs.

In another embodiment, 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 donor of the first population of T cells, the first population of APCs being layered on 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 T cells, the second population of APCs being layered on the first gas permeable surface, the second population of APCs being greater than the first population of APC.

In another embodiment, 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 another embodiment, the invention provides a modified such asThe method of any preceding paragraph above, wherein the number of APCs in the first population of APCs is about 2.5X10 ⁸ In one embodiment, the number of APCs in the second population of APCs is about 5X 10 ⁸ And each.

In another embodiment, 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 on the first ventilation surface with an average thickness of 2 layers of APCs.

In another embodiment, 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 on the first ventilation surface with an average thickness selected from the range of 4 to 8 APC.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the ratio of the average number of APCs laminated on the first ventilation surface in step (b) to the average number of APCs laminated on the first ventilation surface in step (a) is 2:1.

in another embodiment, 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 selected from the group consisting of exactly or about 1.0X10 ⁶ APC/cm ² To exactly or about 4.5 x 10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 selected from the group consisting of exactly or about 1.5X10 ⁶ APC/cm ² To just or about 3.5X10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 selected from the group consisting of exactly or about 2.0X10 ⁶ APC/cm ² To just or about 3.0X10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 at or about 2.0X10 ⁶ APC/cm ² Is inoculated on the first breathable surface.

In another embodiment, 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 selected from the group consisting of exactly or about 2.5X10 ⁶ APC/cm ² To just or about 7.5 x 10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 selected from the group consisting of exactly or about 3.5X10 ⁶ APC/cm ² To exactly or about 6.0X10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 selected from the group consisting of exactly or about 4.0X10 ⁶ APC/cm ² To exactly or about 5.5X10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 at or about 4.0X10 ⁶ APC/cm ² Is inoculated on the first breathable surface.

In another embodiment, 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 selected from the group consisting of exactly or about 1.0X10 ⁶ APC/cm ² To exactly or about 4.5 x 10 ⁶ APC/cm ² Inoculating a first gas permeable surface with a density in the range of (a) in step (b) the second population of APCs is selected from exactly or about 2.5x10 ⁶ APC/cm ² To just or about 7.5 x 10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable, wherein, in step (a), the first population of APCs is selected from the group consisting of exactly or about 1.5X10 ⁶ APC/cm ² To just or about 3.5X10 ⁶ APC/cm ² Inoculating a first gas permeable surface with a density in the range of (a), in step (b), the second population of APCs being selected from exactly or about 3.5X10 s ⁶ APC/cm ² To exactly or about 6.0X10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 selected from the group consisting of exactly or about 2.0X10 ⁶ APC/cm ² To just or about 3.0X10 ⁶ APC/cm ² Inoculating a first gas permeable surface with a density in the range of (a) in step (b) the second population of APCs is selected from exactly or about 4.0x10 ⁶ APC/cm ² To exactly or about 5.5X10 ⁶ APC/cm ² Is inoculated on the first gas-permeable surface.

In another embodiment, 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 at or about 2.0X10 ⁶ APC/cm ² Is inoculated onto the first air-permeable surface at a density of exactly or about 4.0X10 s in the second population of APCs in step (b) ⁶ APC/cm ² Is inoculated on the first breathable surface.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the APCs are Peripheral Blood Mononuclear Cells (PBMCs).

In another embodiment, 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 another embodiment, 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 another embodiment, 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 another embodiment, 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 another embodiment, 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 another embodiment, 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 product from a blood cell isolation of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is isolated from whole blood or blood cell isolation products of the donor by positively or negatively selecting the T cell phenotype.

In another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein the T cell phenotype is cd3+ and cd45+.

In another embodiment, 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 population of T cells. In another embodiment, the T cells are isolated from NK cells in the first T cell population by removing CD3-cd56+ cells from the first T cell population. In another embodiment, the CD3-cd56+ cells are removed from the first T cell population by subjecting the first T cell population to cell sorting using a gating strategy that removes the CD3-cd56+ cell fraction and recovers the negative fraction. In another embodiment, the foregoing method is used to expand T cells in a first T cell population characterized by a high percentage of NK cells. In another embodiment, the foregoing method is used to expand T cells in a first T cell population characterized by a high percentage of CD3-CD56+ cells. In another embodiment, the foregoing method is used to expand T cells in tumor tissue characterized by the presence of high numbers of NK cells. In another embodiment, the foregoing method is used to expand T cells in tumor tissue characterized by high numbers of CD3-CD56+ cells. In another embodiment, the foregoing method is used to expand T cells in tumor tissue obtained from a patient suffering from a tumor characterized by the presence of high numbers of NK cells. In another embodiment, the foregoing method is used to expand T cells in tumor tissue obtained from a patient suffering from a tumor characterized by the presence of high numbers of CD3-CD56+ cells. In another embodiment, the foregoing method is used to expand T cells in tumor tissue obtained from a patient suffering from ovarian cancer.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the T cells are from the first population of T cells at or about 1 x 10 ⁷ The individual T cells are seeded into a vessel to initiate a primary first expansion culture in the vessel.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first population of T cells is distributed in a plurality of containers, and each container is seeded with exactly or about 1X 10 cells from the first population of T cells ⁷ T cells to initiate a primary first expansion culture in the vessel.

In another embodiment, 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 another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from one or more small biopsy (including, for example, a perforated biopsy), a core biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsy sections (including, for example, a perforated biopsy), a core biopsy, a core needle biopsy, or a fine needle aspirate of tumor tissue of the donor.

In another embodiment, 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 sections (including, for example, a perforated biopsy section), a core biopsy section, a core needle biopsy section, or a fine needle aspirate of tumor tissue of the donor.

In another embodiment, 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 biopsy (including, for example, a perforated biopsy), a core biopsy, a core needle biopsy or a fine needle aspirate of tumor tissue of the donor.

In another embodiment, 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 biopsy (including, for example, a perforated biopsy), a core biopsy, a core needle biopsy or a fine needle aspirate of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from one or more core biopsy sections of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 20 core biopsy sections of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 10 core biopsy sections of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 core biopsy of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core biopsy sections of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from one or more fine needle aspirates of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 20 fine needle aspirates of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 10 fine needle aspirates of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 fine needle aspirates of the tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fine needle aspirates of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from one or more small biopsy (including, for example, a punch biopsy) of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 20 small biopsy sections (including, for example, perforated biopsy sections) of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 10 small biopsy sections (including, for example, perforated biopsy sections) of tumor tissue of the donor.

In another embodiment, 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 biopsy (including, for example, a perforated biopsy) of tumor tissue of the donor.

In another embodiment, 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 biopsy (including, for example, a punch biopsy) of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from one or more core needle biopsy sections of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 20 core needle biopsy sections of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1 to 10 core needle biopsy sections of tumor tissue of the donor.

In another embodiment, the invention provides a modified method as described in any preceding paragraph 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 core needle biopsy.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the first T cell population is obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 core needle biopsy of tumor tissue of the donor.

In another embodiment, the invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: i) Obtaining and/or receiving a first population of TILs from a tumor sample obtained from a tumor of a subject by culturing the tumor sample in a first cell culture medium comprising IL-2 for about 3 days; (ii) Performing an initial first amplification by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs) to produce a second population of TILs, wherein the initial first amplification is performed in a vessel comprising a first gas permeable surface area, the initial first amplification being performed for a first period of time 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) 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 to produce a third TIL population, 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 TIL population, the third TIL population being a therapeutic TIL population, the rapid second amplification being performed in a container comprising a second gas permeable surface area; (iv) collecting the therapeutic TIL population obtained from step (iii); and (v) transferring the collected TIL population from step (iv) to an infusion bag.

In another embodiment, the invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (i) Obtaining and/or receiving a first population of TILs from a tumor sample obtained from a tumor of a subject by culturing the tumor sample in a first cell culture medium comprising IL-2 for about 3 days; (ii) Performing an initial first amplification by culturing a first population of TILs in a second cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs) to produce a second population of TILs, wherein the initial first amplification is 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) 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 to produce 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; and (iv) collecting the therapeutic TIL population obtained from step (iii).

In another embodiment, 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 medium is bottled in 2 or more than 2 subcultures, and each subculture is supplemented with an additional amount of the third medium and cultured for about 6 days.

In another embodiment, 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 medium is bottled in 2 or more than 2 subcultures, and each subculture is supplemented with a fourth medium comprising IL-2 and cultured for about 6 days.

In another embodiment, the invention provides a modified method as described in any preceding paragraph as applicable above, wherein the culture medium is bottled into at most 5 subcultures after day 5 of the second period of time.

In another embodiment, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, wherein all steps of the method are completed within about 22 days.

In another embodiment, the invention provides a method of expanding T cells comprising: (i) Performing an initial first expansion of a first T cell population by culturing the first T cell population from a tumor sample obtained from one or more small, core, or needle biopsy from a donor tumor to grow the first T cell population and prepare for activation of the first T cell population; (ii) After the activation of the first T cell population prepared in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population such that the first T cell population grows and the activation of the first T cell population is enhanced to obtain a second T cell population; and (iv) collecting the second T cell population. In some embodiments, the tumor sample is obtained from a plurality of core biopsy sections. In some embodiments, the plurality of core biopsy slices is selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10 core biopsy slices.

In some embodiments, the invention provides a modified method as described in any of the preceding paragraphs as applicable above, 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, miltenyi Biotec, ornithogen, calif.). In some embodiments, the tumor is placed into a tumor dissociating enzyme mixture that may include more than one dissociating (digesting) enzyme, such as, but not limited to collagenases (including collagenases of any blend or type), accutase ^TM 、Accumax ^TM Hyaluronidase, neutral protease (dispase), chymosin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (chain protease), deoxyribonuclease I (dnase), trypsin inhibitor, any other dissociation or proteinA lytic enzyme, and any combination thereof. In other embodiments, the tumor is placed in a tumor dissociating enzyme mixture comprising collagenase (including collagenase of any blend or type), neutral protease (dispase), and dnase I (dnase).

Pharmaceutical compositions, dosages and dosing regimens

In one embodiment, the TIL, MILs, or PBLs amplified and/or genetically modified (including TIL, MILs, or PBLs genetically modified to express CCR) using the methods of the present disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, 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 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.

Any suitable dose of TIL may be administered. In some embodiments, about 2.3X10 are administered ¹⁰ To about 13.7X10 ¹⁰ TIL, average about 7.8X10 ¹⁰ The term TIL is especially used if the cancer is melanoma. In one embodiment, about 1.2X10 is administered ¹⁰ Up to about 4.3X10 ¹⁰ And TIL. In some embodiments, about 3 x 10 is administered ¹⁰ Up to about 12X 10 ¹⁰ And TIL. In some embodiments, about 4 x 10 is administered ¹⁰ Up to about 10X 10 ¹⁰ And TIL. In some embodiments, about 5 x 10 is administered ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, about 6 x 10 is administered ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, about 7 x 10 is administered ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 2.3X10 ¹⁰ To about 13.7X10 ¹⁰ And each. In some embodiments, the therapeutically effective dose is about 7.8X10 ¹⁰ The TIL is especially melanoma. In some embodiments, the therapeutically effective dose is about 1.2X10 ¹⁰ Up to about 4.3X10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about3×10 ¹⁰ Up to about 12X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 4 x 10 ¹⁰ Up to about 10X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 5 x 10 ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 6 x 10 ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 7 x 10 ¹⁰ Up to about 8X 10 ¹⁰ And TIL.

In some embodiments, the amount of TIL provided in the pharmaceutical compositions of the present invention is about 1X 10 ⁶ 、2×10 ⁶ 、3×10 ⁶ 、4×10 ⁶ 、5×10 ⁶ 、6×10 ⁶ 、7×10 ⁶ 、8×10 ⁶ 、9×10 ⁶ 、1×10 ⁷ 、2×10 ⁷ 、3×10 ⁷ 、4×10 ⁷ 、5×10 ⁷ 、6×10 ⁷ 、7×10 ⁷ 、8×10 ⁷ 、9×10 ⁷ 、1×10 ⁸ 、2×10 ⁸ 、3×10 ⁸ 、4×10 ⁸ 、5×10 ⁸ 、6×10 ⁸ 、7×10 ⁸ 、8×10 ⁸ 、9×10 ⁸ 、1×10 ⁹ 、2×10 ⁹ 、3×10 ⁹ 、4×10 ⁹ 、5×10 ⁹ 、6×10 ⁹ 、7×10 ⁹ 、8×10 ⁹ 、9×10 ⁹ 、1×10 ¹⁰ 、2×10 ¹⁰ 、3×10 ¹⁰ 、4×10 ¹⁰ 、5×10 ¹⁰ 、6×10 ¹⁰ 、7×10 ¹⁰ 、8×10 ¹⁰ 、9×10 ¹⁰ 、1×10 ¹¹ 、2×10 ¹¹ 、3×10 ¹¹ 、4×10 ¹¹ 、5×10 ¹¹ 、6×10 ¹¹ 、7×10 ¹¹ 、8×10 ¹¹ 、9×10 ¹¹ 、1×10 ¹² 、2×10 ¹² 、3×10 ¹² 、4×10 ¹² 、5×10 ¹² 、6×10 ¹² 、7×10 ¹² 、8×10 ¹² 、9×10 ¹² 、1×10 ¹³ 、2×10 ¹³ 、3×10 ¹³ 、4×10 ¹³ 、5×10 ¹³ 、6×10 ¹³ 、7×10 ¹³ 、8×10 ¹³ And 9X 10 ¹³ . In one embodiment, the amount of TIL provided in the pharmaceutical composition of the invention is 1X 10 ⁶ Up to 5X 10 ⁶ 、5×10 ⁶ Up to 1X 10 ⁷ 、1×10 ⁷ Up to 5X 10 ⁷ 、5×10 ⁷ Up to 1X 10 ⁸ 、1×10 ⁸ Up to 5X 10 ⁸ 、5×10 ⁸ Up to 1X 10 ⁹ 、1×10 ⁹ Up to 5X 10 ⁹ 、5×10 ⁹ Up to 1X 10 ¹⁰ 、1×10 ¹⁰ Up to 5X 10 ¹⁰ 、5×10 ¹⁰ Up to 1X 10 ¹¹ 、5×10 ¹¹ Up to 1X 10 ¹² 、1×10 ¹² Up to 5X 10 ¹² And 5X 10 ¹² Up to 1X 10 ¹³ Within a range of (2).

In some embodiments, the concentration of TIL provided in a pharmaceutical composition of the present invention is less than, for example, 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/v or v/v of the pharmaceutical composition.

In some embodiments of the present invention, 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%, 12.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10.10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8.8%, 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/v, w/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 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 of the present invention, in some embodiments, the amount of TIL provided in the pharmaceutical composition 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, 0.003g, 0.03g, 0.02g, 0.001g, 0.008g, 0.0008g, 0.0003g, 0.000008 g, 0.0008g, 0.0003g, 0008g, 0.0008g, 0.0003g, 0.001g, 0008g, 0.0008g, 0.0007g, 0.005g, or the present.

In some embodiments of the present invention, 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 broad dosage range. The exact dosage will depend on the route of administration, the form of administration of the compound, the identity and age of the subject to be treated, the weight of the subject to be treated, and the preference and experience of the attending physician. Clinical established doses of TIL may also be used if appropriate. The amount of pharmaceutical composition administered (e.g., the dose of TIL) using the methods herein will depend on the person or mammal to be treated, the severity of the condition or disease, the rate of administration, the disposition of the active pharmaceutical ingredient, and the prescribing physician's considerations.

In some embodiments, the TIL may be administered in a single dose. The administration may be injection, for example intravenous injection. In some embodiments, the TIL may be administered in multiple doses. Administration may be 1, 2, 3, 4, 5, 6 or more than 6 times per year. The administration may be once a month, once every two weeks, once a week or once every two days. Administration of the TIL may continue as desired.

In some embodiments, an effective dose of TIL is about 1X 10 ⁶ 、2×10 ⁶ 、3×10 ⁶ 、4×10 ⁶ 、5×10 ⁶ 、6×10 ⁶ 、7×10 ⁶ 、8×10 ⁶ 、9×10 ⁶ 、1×10 ⁷ 、2×10 ⁷ 、3×10 ⁷ 、4×10 ⁷ 、5×10 ⁷ 、6×10 ⁷ 、7×10 ⁷ 、8×10 ⁷ 、9×10 ⁷ 、1×10 ⁸ 、2×10 ⁸ 、3×10 ⁸ 、4×10 ⁸ 、5×10 ⁸ 、6×10 ⁸ 、7×10 ⁸ 、8×10 ⁸ 、9×10 ⁸ 、1×10 ⁹ 、2×10 ⁹ 、3×10 ⁹ 、4×10 ⁹ 、5×10 ⁹ 、6×10 ⁹ 、7×10 ⁹ 、8×10 ⁹ 、9×10 ⁹ 、1×10 ¹⁰ 、2×10 ¹⁰ 、3×10 ¹⁰ 、4×10 ¹⁰ 、5×10 ¹⁰ 、6×10 ¹⁰ 、7×10 ¹⁰ 、8×10 ¹⁰ 、9×10 ¹⁰ 、1×10 ¹¹ 、2×10 ¹¹ 、3×10 ¹¹ 、4×10 ¹¹ 、5×10 ¹¹ 、6×10 ¹¹ 、7×10 ¹¹ 、8×10 ¹¹ 、9×10 ¹¹ 、1×10 ¹² 、2×10 ¹² 、3×10 ¹² 、4×10 ¹² 、5×10 ¹² 、6×10 ¹² 、7×10 ¹² 、8×10 ¹² 、9×10 ¹² 、1×10 ¹³ 、2×10 ¹³ 、3×10 ¹³ 、4×10 ¹³ 、5×10 ¹³ 、6×10 ¹³ 、7×10 ¹³ 、8×10 ¹³ And 9X 10 ¹³ . In some embodiments, an effective dose of TIL is 1X 10 ⁶ Up to 5X 10 ⁶ 、5×10 ⁶ Up to 1X 10 ⁷ 、1×10 ⁷ Up to 5X 10 ⁷ 、5×10 ⁷ Up to 1X 10 ⁸ 、1×10 ⁸ Up to 5X 10 ⁸ 、5×10 ⁸ Up to 1X 10 ⁹ 、1×10 ⁹ Up to 5X 10 ⁹ 、5×10 ⁹ Up to 1X 10 ¹⁰ 、1×10 ¹⁰ Up to 5X 10 ¹⁰ 、5×10 ¹⁰ Up to 1X 10 ¹¹ 、5×10 ¹¹ Up to 1X 10 ¹² 、1×10 ¹² Up to 5X 10 ¹² And 5X 10 ¹² Up to 1X 10 ¹³ Within a range of (2).

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.

The effective amount of TIL may be administered in more than a single dose by any acceptable mode of administration of the agent with similar utility, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, by implantation, or by inhalation.

In another embodiment, the invention provides an infusion bag comprising a therapeutic TIL population as described in any of the preceding paragraphs above.

In another embodiment, 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 another embodiment, the invention provides an infusion bag comprising a TIL composition as described in any of the preceding paragraphs above.

In another embodiment, the invention provides a cryopreserved formulation of a therapeutic TIL population as described in any preceding paragraph above.

In another embodiment, 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 another embodiment, the invention provides a modified TIL composition as described in any preceding paragraph above, wherein the cryopreservation medium comprises DMSO.

In another embodiment, the invention provides a modified TIL composition as described in any preceding paragraph above, wherein the cryopreservation medium contains 7 to 10% DMSO.

In another embodiment, the invention provides a cryopreserved formulation of a TIL composition as described in any of the preceding paragraphs above.

In one embodiment, the TIL amplified using the methods of the present disclosure is administered to a patient as a pharmaceutical composition. In one embodiment, 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 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.

Any suitable dose of TIL may be administered. In some embodiments, about 2.3X10 are administered ¹⁰ To about 13.7X10 ¹⁰ TIL, average about 7.8X10 ¹⁰ The term TIL is used specifically if the cancer is NSCLC. In one embodiment, about 1.2X10 is administered ¹⁰ Up to about 4.3X10 ¹⁰ And TIL. In some implementationsIn embodiments, about 3X 10 is administered ¹⁰ Up to about 12X 10 ¹⁰ And TIL. In some embodiments, about 4 x 10 is administered ¹⁰ Up to about 10X 10 ¹⁰ And TIL. In some embodiments, about 5 x 10 is administered ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, about 6 x 10 is administered ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, about 7 x 10 is administered ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 2.3X10 ¹⁰ To about 13.7X10 ¹⁰ And each. In some embodiments, the therapeutically effective dose is about 7.8X10 ¹⁰ The TIL is specifically NSCLC. In some embodiments, the therapeutically effective dose is about 1.2X10 ¹⁰ Up to about 4.3X10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 3 x 10 ¹⁰ Up to about 12X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 4 x 10 ¹⁰ Up to about 10X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 5 x 10 ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 6 x 10 ¹⁰ Up to about 8X 10 ¹⁰ And TIL. In some embodiments, the therapeutically effective dose is about 7 x 10 ¹⁰ Up to about 8X 10 ¹⁰ And TIL.

In some embodiments, the TIL may be administered in a single dose. Administration may be injection, for example intravenous injection. In some embodiments, the TIL may be administered in multiple doses. Administration may be 1, 2, 3, 4, 5, 6 or more than 6 times per year. The administration may be once a month, once every two weeks, once a week or once every two days. Administration of the TIL may continue as desired.

Methods of treating patients

The treatment method begins with initial TIL collection and TIL culture, optionally modified as described herein to express more than one CCR and/or more than one chemokine receptor. Such methods of treatment are well known in the art, e.g., jin et al, J.Immunotherapy 2012,35 (3): 283-292, which is incorporated by reference in its entirety. Embodiments of the methods of treatment are described in all sections below, including examples.

Amplified TILs produced according to the methods described herein, including for example those described in steps A through F above (or as shown, for example, in FIG. 1), have particular uses for treating cancer patients (as described, for example, in Goff et al, J.clinical Oncology,2016,34 (20): 2389-239 and supplements); the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, TIL grows from resected registers of metastatic melanoma 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. Can use 2mm ³ To 3mm ³ Is a single fragment of (a). In some embodiments, 5, 10, 15, 20, 25, or 30 samples per patient are obtained. In some embodiments, 20, 25, or 30 samples per patient are obtained. In some embodiments, 20, 22, 24, 26, or 28 samples per patient are obtained. In some embodiments, 24 samples per patient are obtained. The sample can be placed in a separate well of a 24-well plate and maintained in a growth medium containing high dose of IL-2 (6,000IU/mL)In the medium and monitoring for tumor destruction and/or proliferation of TIL. Any tumor that remains viable cells after treatment can be enzymatically digested into a single cell suspension 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 can be considered reactive if overnight co-culture yields interferon-gamma (IFN-gamma) levels > 200pg/mL and twice background (Goff et al, J Immunother.,2010,33:840-847; incorporated herein by reference in its entirety). In some embodiments, culture with auto-reactivity or sufficient evidence of growth patterns may be selected for a second amplification, including a second amplification sometimes referred to as rapid amplification (REP). In some embodiments, amplified TIL with high auto-reactivity (e.g., high proliferation during the second amplification) is selected for additional second amplification. In some embodiments, TIL with high auto-reactivity is selected for additional second REP amplification.

The cell phenotype of the cryopreserved infusion bag TIL samples can be analyzed by flow cytometry (e.g., flowJo) for the surface markers CD3, CD4, CD8, CCR7, and CD45RA (BD BioSciences), as well as by any of the methods described herein. Serum cytokines were measured using standard enzyme-linked immunosorbent assay techniques. The serum IFN-g elevation was defined as > 100pg/mL and greater than 4 3 baseline level.

In some embodiments, TILs produced by the methods provided herein (e.g., those exemplified herein) provide a clinical benefit that unexpectedly improves TILs. In some embodiments, TILs produced by the methods provided herein (e.g., those exemplified in fig. 1) exhibit increased clinical efficacy as compared to TILs produced by methods other than those described herein (including, e.g., those other than the methods exemplified in fig. 1). In some embodiments, those other than the methods described herein include methods known as process 1C and/or generation 1 (Gen 1). In some embodiments, increasing the therapeutic effect is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by the methods provided herein (e.g., those exemplified in fig. 1) exhibit similar reaction time and safety characteristics compared to TILs produced by methods other than those described herein (including, e.g., those other than the methods exemplified in fig. 1, such as Gen 1 processes).

In some embodiments, IFN-gamma indicates therapeutic efficacy and/or increases clinical efficacy. In some embodiments, IFN- γ in the blood of a TIL treatment subject is indicative of active TIL. In some embodiments, the potency assay for IFN-gamma production is used. IFN-gamma production is another measure of cytotoxic potential. IFN-gamma production may be measured by determining the amount of the cytokine IFN-gamma in blood, serum or ex vivo TIL of a subject treated with TIL prepared by the methods of the invention (including, for example, those described in FIG. 1). In some embodiments, an increase in IFN- γ indicates the therapeutic 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 TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, IFN- γ secretion is increased by a factor of 1 compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, IFN- γ secretion is increased by a factor of 2 compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, IFN- γ secretion is increased 3-fold compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, IFN- γ secretion is increased by a factor of 4 compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, IFN- γ secretion is increased 5-fold compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, IFN-. Gamma.is measured using a Quantikine ELISA kit. In some embodiments, IFN- γ is measured in ex vivo TIL of subjects treated with TIL prepared by the methods of the invention (including, for example, those described in fig. 1). In some embodiments, IFN- γ is measured in the blood of subjects treated with TIL prepared by the methods of the invention (including, for example, those described in fig. 1). In some embodiments, IFN- γ is measured in TIL serum of subjects treated with TIL prepared by the methods of the invention (including, for example, those described in fig. 1).

In some embodiments, TILs prepared by the methods of the invention (including, for example, those described in fig. 1) exhibit increased polyclonality compared to TILs produced by other methods (including those not illustrated in fig. 1, e.g., methods known as the process 1C method). In some embodiments, significantly improved 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 TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 1 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 2 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 10 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 100 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 500 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased 1000-fold compared to untreated patients and/or compared to patients treated with TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1).

The measurement of efficacy may include Disease Control Rate (DCR) and Overall 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 methods of treating diseases. In one embodiment, 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 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, cervical squamous carcinoma and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal-gastric junction cancer, gastric cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC), hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer and pharyngeal cancer), renal cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including grape-membrane melanoma, choriocarcinoma, ciliary melanoma or iris melanoma), pleural mesothelioma (including malignant mesothelioma), ovarian cancer, pancreatic cancer (including ductal adenocarcinoma), penile carcinoma, renal sarcoma, osteosarcoma, esarcoma, thyroid carcinoma, sarcoma, and other carcinoma (including sarcoma of the thyroid gland, sarcoma, and other carcinoma, sarcoma, and carcinoma of the uterus (including the human body, and carcinoma).

In some embodiments, the hyperproliferative disorder is a hematological malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of: 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 a method of treating a patient with cancer, which is a hematological malignancy. In some embodiments, the invention includes a method of treating a patient with cancer, which is a hematological malignancy, using a TIL, MILs, or PBL modified to express more than one CCR. In some embodiments, the invention includes a method of treating a patient with cancer, which is a hematological malignancy, using MILs or PBLs modified to express more than one CCR.

In one embodiment, the cancer is one of the foregoing cancers, including solid tumor cancers and hematological malignancies, that present a recurrence or refractory to treatment with at least one prior therapy (including chemotherapy, radiation therapy, or immunotherapy). In one embodiment, the cancer is one of the foregoing cancers, which exhibits relapse or refractory to treatment by at least two previous therapies, including chemotherapy, radiation therapy, and/or immunotherapy. In one embodiment, the cancer is one of the foregoing cancers, which exhibits relapse or refractory to treatment by at least three previous therapies, including chemotherapy, radiation therapy, and/or immunotherapy.

In some embodiments, the cancer is a microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) cancer. MSI-H and dMMR cancers and tests have thus been described in Kawakami et al, curr. Treat. Options Oncol.2015,16,30, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the invention includes a method of treating a cancer patient with a TIL, MILs, or PBL modified to express more than one CCR, wherein the patient is a human. In some embodiments, the invention includes a method of treating a cancer patient with a TIL, MILs, or PBL modified to express more than one CCR, wherein the patient is a non-human. In some embodiments, the invention includes a method of treating a cancer patient with 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 cancer patient with a TIL, MILs, or PBL modified to express more than one CCR, wherein the patient is a primate, horse, dog, or feline.

In some embodiments, the invention includes a method of treating a patient with cancer that presents a refractory to treatment with a BRAF inhibitor and/or a MEK inhibitor. In some embodiments, the invention includes a method of treating a patient with cancer that is refractory to treatment with a BRAF inhibitor selected from the group consisting of vemurafenib (vemurafenib), dabrafenib (dabrafenib), encouraging fenib (encorafenib), sorafenib (sorafenib), and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the invention includes a method of treating a patient with cancer that is refractory to treatment with a MEK inhibitor selected from the group consisting of trametinib (trametinib), cobimetinib (cobimetinib), bi Ni tinib (binimetinib), semetinib (selumetinib), pimassitinib (pimasetinib), refametinib (refametinib), and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the invention includes a method of treating a patient with cancer that is refractory to treatment with a BRAF inhibitor selected from the group consisting of vemurafenib, dabrafenib, enofenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof, and a MEK inhibitor selected from the group consisting of trametinib, cobicitinib, bi Niti ni, semtinib, pid Ma Siti ni, and refatinib, and pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is pediatric cancer.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the cancer is uveal melanoma.

In some embodiments, the invention includes a method of treating a cancer patient, 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 with cancer, wherein the pediatric cancer is a neublastic tumor.

In some embodiments, the invention includes a method of treating a patient with cancer, wherein the pediatric cancer is a sarcoma.

In some embodiments, the invention includes a method of treating a cancer patient, wherein the sarcoma is osteosarcoma.

In some embodiments, the invention includes a method of treating a cancer patient, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the invention includes a method of treating a cancer patient, wherein the soft tissue sarcoma is rhabdomyosarcoma, ewing's sarcoma, or primitive neuroectodermal tumor.

In some embodiments, the invention includes a method of treating a cancer patient, wherein the pediatric cancer is a Central Nervous System (CNS) -related cancer. In some embodiments, pediatric cancers present a refractory to treatment with chemotherapy. In some embodiments, pediatric cancers present a refractory to treatment with radiation therapy. In some embodiments, the pediatric cancer presents a refractory to treatment with denotuximab (dinutuximab).

In some embodiments, the invention includes a method of treating a cancer patient, wherein the CNS-related cancer is a neuroblastoma, pineal blastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein can be used in methods of treating cancer that is refractory or resistant to prior treatment with anti-PD-1 or anti-PD-L1 antibodies. 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 antibody. In some embodiments, the patient exhibits a prior response against PD-1 or an anti-PD-L1 antibody, followed by progression of the patient's cancer. In some embodiments, the cancer presents a refractory to the combination of an anti-CTLA-4 antibody and/or an anti-PD-1 or an anti-PD-L1 antibody with at least one chemotherapeutic agent. In some embodiments, the previous chemotherapeutic agent is carboplatin, paclitaxel, 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, gemcitabine, and a taxane (including, for example, paclitaxel, 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 is from a patient as described elsewhere herein with a cancer that expresses PD-L1 at a tumor fraction (TPS) of < 1%.

In some embodiments, NSCLC presents a refractory to a combination therapy comprising an anti-PD-1 or 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

a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine and a taxane including, for example, paclitaxel, docetaxel or albumin-bound paclitaxel.

In some embodiments, NSCLC presents a refractory to a combination therapy comprising an anti-PD-1 or 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 patient with NSCLC is untreated (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 no longer currently treated with the chemotherapeutic agent. In some embodiments, the patient with NSCLC does not receive anti-PD-1/PD-L1. In some embodiments, the NSCLC patient has low expression of PD-L1. In some embodiments, NSCLC in a NSCLC patient is untreated or is post-chemotherapeutic but not anti-PD-1/PD-L1. In some embodiments, a patient with NSCLC is untreated or is treated with a chemotherapeutic agent but is not treated with anti-PD-1/PD-L1 and has low expression of PD-L1. In some embodiments, the NSCLC patient has a large tumor at baseline. In some embodiments, the subject has a large tumor at baseline and has low expression of PD-L1. 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 is at baseline With large tumors and no detectable PD-L1 expression. In some embodiments, NSCLC in a NSCLC patient has not been treated or after chemotherapy (after a chemotherapeutic agent) but has not been anti-PD-1/PD-L1 and has low expression of PD-L1 and/or has a large tumor at baseline. In some embodiments, a large tumor is indicated when the maximum tumor diameter measured in a transverse or coronal plane is greater than 7 cm. In some embodiments, a large tumor is indicated when the minor axis diameter of the swollen lymph node is 20mm or greater than 20 mm. In some embodiments, the chemotherapeutic agent comprises a standard care therapeutic agent for NSCLC.

In some embodiments, PD-L1 expression is determined from a tumor proportion score. In some embodiments, a subject with refractory NSCLC tumor has a tumor fraction of <1% (TPS). 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 the tumor fraction is 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 the tumor fraction is determined prior to the anti-PD-L1 antibody treatment.

In some embodiments, TILs prepared by the methods of the invention (including, for example, those described in fig. 1) exhibit increased polyclonality compared to TILs produced by other methods (including those not illustrated in fig. 1, e.g., methods known as the process 1C method). In some embodiments, significantly improved polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy of a 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 TIL prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 1 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 2 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 10 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 100 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). In some embodiments, the polyclonality is increased by a factor of 500 compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than the methods embodied in fig. 1). 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, those other than the methods embodied in fig. 1).

In some embodiments, PD-L1 expression is determined by tumor proportion score using one or more of the test methods as described herein. In some embodiments, the subject or patient with NSCLC tumor has a <1% tumor fraction (TPS). In some embodiments, the NSCLC tumor has ≡1% TPS. In some embodiments, a subject or patient having NSCLC has been previously treated with an anti-PD-1 and/or anti-PD-L1 antibody, and the tumor fraction is determined prior to the anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject or patient having NSCLC has been previously treated with an anti-PD-L1 antibody, and the tumor fraction is determined prior to the anti-PD-L1 antibody treatment. In some embodiments, a subject or patient with refractory or resistant NSCLC tumor has a <1% tumor fraction (TPS). 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 the tumor fraction is determined prior to the anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, a subject or patient with refractory or resistant NSCLC has been previously treated with an anti-PD-L1 antibody, and the tumor fraction is determined prior to the anti-PD-L1 antibody treatment.

In some embodiments, NSCLC is NSCLC exhibiting a tumor fraction (TPS) or a percentage of surviving tumor cells exhibiting partial or complete membrane staining of PD-L1 protein of any intensity collected from a patient prior to anti-PD-1 or anti-PD-L1 therapy of less than 1% (TPS < 1%). In one embodiment, NSCLC is an NSCLC exhibiting a ratio selected from <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% TPS. In one embodiment, the NSCLC is a NSCLC exhibiting 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 one embodiment, the NSCLC is NSCLC exhibiting between 0% and 1% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.9% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.8% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.7% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.6% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.5% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.4% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.3% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.2% TPS. In one embodiment, the NSCLC is NSCLC exhibiting between 0% and 0.1% TPS. TPS may be measured by methods known in the art, such as those described in Hirsch, et al j. Thorac. Oncol.2017,12,208-222 or those used to determine TPS prior to treatment with 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 an 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 than 99%. In some embodiments, full film staining comprises about 100% film staining.

In some embodiments, the PD-L1 test may involve measuring the level of PD-L1 in the serum of a patient. In these embodiments, the uncertainty of PD-L1 removal of tumor heterogeneity and patient discomfort of consecutive biopsy in patient serum is measured.

In some embodiments, an elevated level of soluble PD-L1 as compared to baseline or standard is associated with a poorer prognosis for NSCLC. See, e.g., okuma et al Clinical Lung Cancer,2018,19,410-417; vecchiaralli et al, oncostarget, 2018,9,17554-17563. In some embodiments, PD-L1 is an exosome PD-L1. In some embodiments, PD-L1 is expressed on circulating tumor cells.

In one embodiment, the invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a tumor-infiltrating lymphocyte (TIL) population to a subject or patient in need of treatment, wherein the subject or patient has at least one of:

i. A pre-determined tumor fraction of PD-L1 (TPS) <1%,

TPS fraction of PD-L1 is 1% to 49%, or

The absence of more than one driving mutation is determined beforehand,

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, crebp 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, the method comprising:

(a) Obtaining and/or receiving a first population of TILs from a tumor resected by 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 the closed system;

(c) Generating a second population of TILs by culturing the first population of TILs in a cell culture medium comprising IL-2 to perform a first amplification, wherein the first amplification is performed in a closed vessel providing a first gas-permeable surface area, the first amplification is performed for about 3 to 14 days to obtain the second population of TILs, the second population of TILs being at least 50 times higher in number than the first population of TILs, the transition from step (b) to step (c) occurring without opening the system;

(d) Performing a second amplification by supplementing cell culture medium of a second TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs) to produce a third TIL population, wherein the second amplification is performed for about 7 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) occurring without opening the system;

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

(f) Transferring the collected population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to step (f) occurs without opening the system;

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

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

In one embodiment, the invention provides a method of treating non-small cell lung cancer (NSCLC) by administering a tumor-infiltrating lymphocyte (TIL) population to a patient in need of treatment, wherein the method comprises:

(a) Testing the patient for PD-L1 expression of tumors and tumor proportion fraction (TPS) of PD-L1;

(b) The test patient does not have 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, crebp 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 fraction of about 1% to about 49% and determining that the patient also does not have a driving mutation;

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

(e) Adding a first TIL population to the closed system;

(f) Generating a second population of TILs by culturing the first population of TILs in a cell culture medium comprising IL-2 to perform a first amplification, wherein the first amplification is performed in a closed vessel providing a first gas-permeable surface area, the first amplification is performed for about 3 to 14 days to obtain the second population of TILs, the second population of TILs being at least 50 times higher in number than the first population of TILs, the transition from step (e) to step (f) occurring without opening the system;

(g) Generating a third population of TILs by supplementing cell culture media of the second population of TILs with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs) for a second amplification, 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) occurring without opening the system;

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

(i) Transferring the collected population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to step (f) occurs without opening the system;

(j) Cryopreserving the infusion bag from step (f) containing the collected TIL population using a cryopreservation process; and

(k) Administering a therapeutically effective dose of the third TIL population from the infusion bag of step (g) to a subject or patient.

(c) Determining that the patient has a PD-L1 TPS fraction of less than about 1% and determining that the patient also does not have a driving mutation;

(e) Adding a first TIL population to the closed system;

(b) The test patient does not have 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 the closed system;

In another embodiment, the invention provides a method for treating a cancer subject, the method comprising administering to the subject a therapeutically effective dose of a therapeutic TIL population as described herein.

In another embodiment, the invention provides a method for treating a subject with cancer, the method comprising administering to the subject a therapeutically effective dose of a TIL composition as described herein.

In another embodiment, the invention provides a modified method for treating a cancer subject as described herein, wherein a non-myeloablative lymphocyte depletion regimen is administered to the subject prior to separate administration of a therapeutically effective dose of a therapeutic TIL population and a TIL composition, as described herein. Suitable non-myeloablative lymphocyte depletion protocols are described herein.

In another embodiment, the invention provides a modified subjectThe methods described herein for treating a subject with cancer, a non-myeloablative lymphocyte depletion regimen comprising administering a dose of 60mg/m ² Cyclophosphamide per day for two days and then administered at a dose of 25mg/m ² Five days total of steps per day of fludarabine.

In one embodiment, the invention provides a method of treating a cancer subject as described herein using a TIL, MILs, or PBL as described herein, optionally genetically modified to express CCR and/or chemokine receptors as described herein, the method further comprising administering a pharmaceutical composition comprising apaminomab ¹³¹ I or a variant, fragment or biological analogue thereof. Apamimumab- ¹³¹ I is also known as IOMAB-ACT, an anti-CD 45 antibody available from Actinium Pharmaceuticals, inc.

In one embodiment, the invention provides a method of treating a cancer subject as described herein using a TIL, MIL, or PBL as described herein, optionally genetically modified to express CCR and/or chemokine receptors as described herein, the method further comprising the step of administering a lymphocyte depletion regimen comprising alemtuzumab (alemtuzumab) or a variant, fragment, or biological analog thereof. Alemtuzumab, also known as LEMTRADA, is available from Sanofi, inc.

In one embodiment, the invention provides a method of treating a cancer subject as described herein using a TIL, MILs, or PBL as described herein, wherein the step of replacing the IL-2 regimen of the subject with a CCR as described herein, such that the IL-2 regimen is not administered to the subject in combination with the TIL, MILs, or PBL therapy. In one embodiment, the invention provides a method of treating a cancer subject as described herein using a TIL, MILs, or PBL as described herein, wherein the IL-2 regimen is not administered to the subject in combination with a TIL, MILs, or PBL therapy. In one embodiment, the invention provides a method of treating a cancer subject as described herein using a TIL, MILs, or PBL as described herein, wherein the TIL, MILs, or PBL is modified to express CCR, and no IL-2 regimen is administered to the subject in combination with the TIL, MILs, or PBL therapy. In one embodiment, the invention provides a method of treating a cancer subject as described herein using a TIL, MILs, or PBL as described herein, wherein CCR and/or chemokine receptors as described herein having an IL-2R intracellular domain (including IL-2rβ and IL-2rγ domains) are used, wherein no IL-2 regimen is administered to the subject in combination with a TIL, MILs, or PBL therapy.

In another embodiment, the invention provides a modified method for treating a subject with cancer as described herein, further comprising the step of beginning the treatment of the subject with a high dose IL-2 regimen the next day after the administration of TIL cells to the subject.

In another embodiment, the invention provides a modified method for treating a cancer subject as described herein, wherein the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg every eight hours with 15 minutes bolus intravenous infusion until tolerized.

In another embodiment, the invention provides a method for treating a subject with cancer, which is a solid tumor.

In another embodiment, the invention provides a method for treating a subject with cancer, the cancer being 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, or renal cell carcinoma.

In another embodiment, the invention provides a method for treating a subject with cancer, such as melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In another embodiment, the invention provides a method for treating a subject with cancer, the cancer being melanoma.

In another embodiment, the invention provides a method for treating a subject with cancer, which is HNSCC.

In another embodiment, the invention provides a method for treating a subject with cancer, the cancer being cervical cancer.

In another embodiment, the invention provides a method for treating a subject with cancer, the cancer being NSCLC.

In another embodiment, the invention provides a method for treating a subject with cancer, which is glioblastoma (including GBM).

In another embodiment, the invention provides a method for treating a subject with cancer, the cancer being gastrointestinal cancer.

In another embodiment, the invention provides a method for treating a subject with cancer, which is a high mutation cancer.

In another embodiment, the invention provides a method for treating a subject with cancer, which is pediatric hypermutated cancer.

In another embodiment, the invention provides a therapeutic TIL population as described herein for use in a method of treating a cancer subject, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

In another embodiment, the invention provides a TIL composition described herein for use in a method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In another embodiment, 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 another embodiment, the invention provides a modified therapeutic TIL population or TIL composition as described herein, the non-myeloablative lymphocyte depletion regimen comprising administering a dose of 60mg/m ² Cyclophosphamide per day for two days and then administered at a dose of 25mg/m ² Five days total of steps per day of fludarabine.

In another embodiment, the invention provides a modified therapeutic TIL population or TIL composition described herein to further comprise the step of starting the treatment of the patient with the high dose IL-2 regimen the next day after administration of the TIL cells to the patient.

In another embodiment, the invention provides a modified therapeutic TIL population or TIL composition described herein, wherein the high dose IL-2 regimen comprises administration of 600,000 or 720,000IU/kg every eight hours at 15 minutes of bolus intravenous infusion until tolerized.

In another embodiment, the invention provides a modified therapeutic TIL population or TIL composition as described herein, the cancer being a solid tumor.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, the cancer being 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, or renal cell carcinoma.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, the cancer being melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, the cancer being melanoma.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, the cancer being HNSCC.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, which is cervical cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, the cancer being NSCLC.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, the cancer being glioblastoma.

In another embodiment, the invention provides a therapeutic population of TILs or a composition of TILs for use in treating cancer, the cancer being gastrointestinal cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, which is a high mutation cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition for use in treating cancer, which is pediatric hypermutated cancer.

In some embodiments, the cancer is a high mutation cancer or a high mutation cancer phenotype. Highly mutated cancers are widely described in Campbell et al, cell 2017,171,1042-1056; the entire contents of which are incorporated herein by reference in their entirety for all purposes). In some embodiments, the high mutation tumor comprises between 9 and 10 mutations per million bases (Mb). In some embodiments, the pediatric hypermutated tumor comprises 9.91 mutations per megabase (Mb). In some embodiments, an adult high mutant tumor comprises 9 mutations per million bases (Mb). In some embodiments, the enhanced hypermutated tumor comprises between 10 and 100 mutations per million bases (Mb). In some embodiments, the enhanced pediatric hypermutated tumor comprises between 10 and 100 mutations per million bases (Mb). In some embodiments, the enhanced adult hypermutated tumor comprises between 10 and 100 mutations per million bases (Mb). In some embodiments, an ultra-hypermutated (ultra-hypermutated) tumor comprises greater than 100 mutations per megabase (Mb). In some embodiments, the pediatric ultra-high mutant tumor comprises greater than 100 mutations per million bases (Mb). In some embodiments, an adult ultra-high mutant tumor comprises greater than 100 mutations per million bases (Mb).

In some embodiments, the high mutant tumor has a mutation in the replication repair pathway. In some embodiments, the high mutant tumor has a mutation in replication repair associated with a DNA polymerase. In some embodiments, the high mutant tumor has a slight Wei Xingti instability. In some embodiments, the ultra-high mutant tumor has a mutation in replication repair associated with DNA polymerase and has a slight Wei Xingti instability. In some embodiments, the hypermutation of the tumor is associated with a response to an immune checkpoint inhibitor. In some embodiments, the hypermutated tumor is resistant to immune checkpoint inhibitor treatment. In some embodiments, high mutant tumors may be treated using the TIL of the present invention. In some embodiments, the high mutation of the tumor is caused by environmental factors (external exposure). For example, UV light can be the cause of high numbers of mutations in malignant melanoma (see, e.g., pfeifer, et al Mutat Res.2005,571,19-31; sage, photochem. Photobiol.1993,57, 163-174). In some embodiments, for lung and throat tumors, high mutations in the tumor can be caused by more than 60 carcinogens in cigarette smoke mist, as well as other tumors due to direct mutagen exposure (see, e.g., plaasance et al, nature 2010,463,184-190). In some embodiments, the high mutation of the tumor is caused by an apolipoprotein B mRNA editing enzyme catalyzed polypeptide-like (apodec) family member disorder, which has been shown to result in increased levels of C-to-T conversion in a variety of cancers (see, e.g., roberts et al, nat. Genet.2013,45, 970-976). In some embodiments, the high mutation of the tumor is caused by defective DNA replication repair due to a mutation that disrupts debug (debug) by the primary replicases Pol3 and Pold 1. In some embodiments, the hypermutation of the tumor is caused by a defect in DNA mismatch repair, which is associated with a hypermutation in colorectal, endometrial, and other cancers (see, e.g., kandoth et al, nature 2013,497,67-73; muzny et al, nature 2012,487,330-337). In some embodiments, DNA replication repair mutations are also found in cancer diathesis syndrome (cancer predisposition syndrome), such as constitutional or biallelic mismatch repair deficiency (CMMRD), lynch syndrome, and polymerase debug associated polyposis (PPAP).

In one embodiment, the invention includes a method of treating cancer with a population of TILs, the cancer being a high mutation cancer. In one embodiment, the invention includes a method of treating cancer with a population of TILs, the cancer being an enhanced high mutation cancer. In one embodiment, the invention includes a method of treating cancer with a population of TILs, the cancer being an ultra-high mutation cancer.

In another embodiment, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of a therapeutic TIL population.

In another embodiment, 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.

In another embodiment, the invention provides a method of treating cancer in a patient comprising administering to the subject a non-myeloablative lymphocyte depletion regimen and then 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. Methods of treating cancer based on driver mutations

As used herein, the phrases "driving mutation" and/or "actionable mutation" and/or "oncogenic driving mutation" refer to a mutation that is generally considered an oncogenic driving factor (i.e., a cancer driving factor or a cancer inducer). The presence of more than one of these mutations has traditionally been used as a target for targeted therapies. Typically, driving mutations into treatment via the target therapeutic moiety includes, for example, examination and/or analysis of Tyrosine Kinase Inhibitors (TKIs). Such driving mutations may in some embodiments affect/affect the response to the first line therapeutic treatment. The TIL treatment methods and compositions described herein are effective in treating patients or subjects, whether or not such driving mutations are present. Such driver mutations can be tested and determined by any method known in the art, including whole-exome sequencing or detection methods targeting a particular driver mutation.

In some embodiments, the cancer is a cancer that exhibits the presence or absence of more than one driving mutation. In some embodiments, the cancer exhibits the presence of more than one driving mutation. In some embodiments, the cancer exhibits the absence of more than one driving mutation. In some embodiments, the cancer is analyzed for the absence or presence of one or more driving mutations. In some embodiments, one or more driving mutations are absent. In some embodiments, the cancer treatment is independent of the presence or absence of more than one driving mutation. In some embodiments, the cancer exhibits one or more driving mutations selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20, KRAS mutations, BRAF V600E mutations, BRAF V600K mutations, BRAF V600 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 splice 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, FBXW7 mutations, CCND3 mutations and GNA11 mutations. In some embodiments, the cancer exhibits <1% PD-L1TPS and the absence of more than one driving mutation is pre-determined.

In some embodiments, the cancer is a cancer that is not suitable for treatment by: EGFR inhibitors, BRAF inhibitors, ALK inhibitors, C-Ros inhibitors, RET inhibitors, ERBB2 inhibitors, BRCA inhibitors, MAP2K1 inhibitors, PIK3CA inhibitors, CDKN2A inhibitors, PTEN inhibitors, UMD inhibitors, NRAS inhibitors, KRAS inhibitors, NF1 inhibitors, MET inhibitors, TP53 inhibitors, CREBBP inhibitors, KMT2C inhibitors, KMT2D mutations, ARID1A mutations, RB1 inhibitors, ATM inhibitors, SETD2 inhibitors, FLT3 inhibitors, PTPN11 inhibitors, FGFR1 inhibitors, EP300 inhibitors, MYC inhibitors, EZH2 inhibitors, JAK2 inhibitors, xw7 inhibitors, CCND3 inhibitors, and GNA11 inhibitors.

In some embodiments, the cancer exhibits <1% PD-L1TPS and is a cancer unsuitable for treatment by: EGFR inhibitors, BRAF inhibitors, ALK inhibitors, C-Ros inhibitors, RET inhibitors, ERBB2 inhibitors, BRCA inhibitors, MAP2K1 inhibitors, PIK3CA inhibitors, CDKN2A inhibitors, PTEN inhibitors, UMD inhibitors, NRAS inhibitors, KRAS inhibitors, NF1 inhibitors, MET inhibitors, TP53 inhibitors, CREBBP inhibitors, KMT2C inhibitors, KMT2D mutations, ARID1A mutations, RB1 inhibitors, ATM inhibitors, SETD2 inhibitors, FLT3 inhibitors, PTPN11 inhibitors, FGFR1 inhibitors, EP300 inhibitors, MYC inhibitors, EZH2 inhibitors, JAK2 inhibitors, xw7 inhibitors, CCND3 inhibitors, and GNA11 inhibitors.

In some embodiments, the cancer is NSCLC and the EGFR mutation results in the tumor transitioning from NSCLC to Small Cell Lung Cancer (SCLC).

In some embodiments, the cancer (or a biopsy thereof) exhibits a high tumor mutational burden (high TMB; >10 mut/kb) and/or microsatellite high instability (MSI-high). In some embodiments, the cancer (or a biopsy thereof) exhibits a high tumor mutation burden (high TMB; >10 mut/kb). In some embodiments, the cancer (or a biopsy thereof) exhibits microsatellite high instability (MSI-high). Methods and systems for assessing tumor mutational burden are known in the art. Exemplary disclosures of such methods and systems can be found in U.S. patent No. 9,792,403, U.S. patent application publication No. US 2018/0363066 A1, international patent application publication nos. WO 2013/070634 A1 and WO 2018/106884 A1 and Metzker, nature biotechnol. Rev.2010,11,31-46, each of which is incorporated herein by reference in its entirety.

In some embodiments, EGFR mutations include, for example, but are not limited to, T790M, ex19Del, L858R, exon 20 insertion, delE709-T710insD, I744_k745insKIPVAI, K745_e746insTPVAI K, E709X, E709K, E a, exon 18, except, G719X, G719S, L861, G719 768I, L747P, A763_764insFQEA, D770_n771insNPG, a763_764insFQEA, P772_h773insDNP exon 20 insertion, H773_v774insNPH exon 20 insertion, S768I, D N771insSVD, V769_d770 asv, p.k745_e insipva, p.k745_e746 instpik, p.744_k 745 inspvai, D771 insp 770_fai, D771 insp, D7720, D774 insp, P773_fag, P773 insg, P773 insb, and qK 772 (EGFR repeats/773/b). In some embodiments, the EGFR mutation is selected from the group consisting of: T790M, ex19Del, L858R, exon 20 insertion, delE709-T710insD, I744_K745insKIPVAI, K745_E746insTPVAIK, E709X, E ins 709K, E709A, exon 18, except, G719X, G719A, G ins719S, L861, Q, S768I, L747P, A InQEA, D770_N771insNPG, A763_764insFQEA, P772_H773insDNP exon 20 insertion, H773_V774insNPH exon 20 insertion, S I, D770_N771insSVD, V769_D770InsASV, p.K745_E746insIPVAIK, p.K745_E insTPIK, p.I744_K insKIAI, D770_N771insNPG, P772_H2, P773 InP and EGFR 764 InP (EGFR 764 InsFQEA) repeats of the EGFR.7620 domain.

In some embodiments, EGFR mutations are double mutations, including but not limited to L858R/T790M, ex Del/T790M, G719X/L861Q, G719X/S768I (or S768I/G719X), S768I/L858R, L R/E709A and/or E746_T751 dellinsA+T 790M. In some embodiments, the EGFR mutation is a double mutation selected from the group consisting of: L858R/T790M, ex Del/T790M, G719X/L861Q, G719X/S768I (or S768I/G719X), S768I/L858R, L858R/E709A and E746_T751delinsA+T790M. Additional properties and methods regarding EGFR mutations are provided in international patent application publication No. WO 2010/020618 A1 (which is incorporated herein by reference in its entirety).

In some embodiments, ALK mutations include, but are not limited to, EML4-ALK variant 1 (AB 274722.1; BAF 73611.1), EML4-ALK variant 2 (AB 275889.1; BAF 73612.1), EML4-ALK variant 3a (AB 374361.1; BAG 55003.1), EML4-ALK variant 3B (AB 374362.1; BAG 55004.1), EML4-ALK variant 4 (AB 374363.1; BAG 75147.1), EML4-ALK variant 5a (AB 374364.1; BAG 75148.1), EML4-ALK variant 5B (AB 374365.1; BAG 75149.1), EML4-ALK variant 6 (AB 462411.1; BAH 57335.1), EML4-ALK variant 7 (AB 462412.1; BAH 57336.1), KIF5B-ALK (AB 462413.1; BAH 57337.1), NPM-ALK, TPM3-ALK, GXL-ALK, TETC-ALK, TFGS-ALK, A-11C-ALK variant 5B (AB 374365.1; BAG 75149.1), EML4-ALK variant 6 (AB-ALK), EML4-ALK variant 7 (AB-ALK-35; BAH 858) and so forth, (see, e.g., PRL-35, GL-ALK-35, GL-ALK-8, and so forth, see, e.g., cell-8, and so forth. Furthermore, one of skill in the art will appreciate that ALK kinase variants may be generated depending on the particular fusion event between the ALK kinase and its fusion partner (e.g., EML4 may fuse at least exons 2, 6a, 6b, 13, 14, and/or 15), as described, for example, in Horn and Pao, j.clin.oncol.2009,27,4247-4253, the disclosure of which is incorporated herein by reference in its entirety.

Additional examples of ALK mutations are described in U.S. patent nos. 9,018,230 and 9,458,508, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, ROS1 mutations of the invention are ROS1 fusions, wherein a portion of an ROS1 polypeptide (or polynucleotide encoding it) comprising the kinase domain of an ROS1 protein is fused to all or a portion of another polypeptide (or polynucleotide encoding it) the name of the second polypeptide or polynucleotide is named in the fusion. In some embodiments, the ROS1 mutation is determined as an ROS1 fusion protein (e.g., by IHC) and/or ROS fusion gene (e.g., by FISH) and/or ROS1mRNA (e.g., by qRT-PCR), preferably indicating that the ROS1 fusion protein is selected from the group consisting of: SLC34A2-ROS1 (SLC 34A2 exons 13del2046 and 4 fused to ROS1 exons 32 and 34), CD74-ROS1 (CD 74 exon 6 fused to ROS1 exons 32 and 34), EZR-ROS1 (EZR exon 10 fused to ROS1 exon 34), TPM3-ROS1 (TPM 3 exon 8 fused to ROS1 exon 35), LRIG3-ROS1 (LRIG 3 exon 16 fused to ROS1 exon 35), SDC4-ROS1 (SDC 4 exons 2 and 4 fused to ROS1 exons 32 and SDC4 exon 4 fused to ROS1 exon 34), GOPC-ROS1 also referred to FIG-ROS1 (GOPC exon 8 fused to ROS1 exon 35 and GOPC exon 4 fused to ROS1 exon 36) and G2032R also referred to as ROS1 ^G2032R 。

Additional disclosures of ROS1 mutation and ROS fusion have been provided in U.S. patent application publication nos. US 2010/0221737 A1, US 2015/0056193 A1 and US 2010/0143918 A1, and international patent application publication No. WO 2010/093928 A1 (each of which is incorporated herein by reference in its entirety). In some embodiments, the RET mutation is a RET fusion or a point mutation.

In some embodiments, RET point mutations include, but are not limited to, H6650, K666 686 691 694 700 706 713 736 748 750 765 768 768 769 770 771 777 7781, Q781 790 791 791 804 804 804 806 806 806 806 806 806 819 823 flexible 841 833 841 841 844 844 844 848T, 1852 866 876 886 883 883 883 884 886 891 8970, D898 901K, 5904 904C2, K907 908 912 918 918 918L6, a919 921 922 922 930 961 972 982 1009 1017 10416, and M1064T.

In some embodiments, the RET is fused to a fusion partner selected from the group consisting of: BCR, BCR, CLIP 1, KIFSB, CCDC6, PTClex9, NCOA4, TRIM33, ERC1, FGFrIOP, MBD1, RAB61P2, PRKARIA, TRIM24, KTN1, GOLGA5, HOOK3, KIAA1468, TRIM27, AKAP13, FKBP15, SPECCIL, TBL1XR1, CEP55, CUX1, ACBD5, MYH13, PIBF1, KIAA1217 and MPRIP.

Additional disclosure of RET mutations has been provided in U.S. Pat. No. 10035789, which is hereby incorporated by reference in its entirety.

In some embodiments, the BRAF mutation is a BRAF V600E/K mutation. In other embodiments, the BRAF mutation is a non-V600E/K mutation.

In some embodiments, the non-V600E/K BRAF mutation is a kinase activation mutation, a kinase damage mutation, or a kinase unknown mutation, and combinations thereof. In some embodiments, the kinase activating mutation is selected from R4621, 1463S, G464E, G464R, G464V, G466A, G469 8238 is, E586K, F595L, L547Q, L597R, L5975, L597V, A598V, T5987 600E, V R, K601E, 5602D, A728V, and combinations thereof. In some embodiments, the kinase impairment mutation is selected from G466E, G466R, G V, Y472C, K483M, D594A, D594E, D594G, D594H, D594N, D594V, G596R, T599A, 5602A, and combinations thereof. In some embodiments, the kinase unknown mutation is selected from T4401, 5467L, G469E, G469R, G4695, G469V, L584F, L588F, V K6oldelins e, 56051, Q609L, E611Q, and combinations thereof. In some embodiments, the non-V600E/K BRAF mutation is selected from the group consisting of D594, G469, K601E, L597, T599 repeat, L485W, F247L, G466V, BRAF fusion, BRAF-AGAP3 rearrangement, BRAF exon 15 splice variant, and combinations thereof.

In some embodiments, met mutations include point mutations, deliberate mutations, insertional mutations, inversions, aberrant splicing, missense mutations, or gene amplification, which result in increased at least one biological activity of the c-Met protein, tyrosine kinase activity such as improvement, receptor homolog dimerization ligand binding formation, enhancement of body and heterodimer, and the like. Met mutations may be located in any part of the c-Met gene. In one embodiment, the mutation is in the kinase domain of the c-MET protein encoded by the c-MET gene. In some embodiments, the c-Met mutation is a point mutation at N375, V13, V923, R175, V136, L229, S323, R988, S1058/T1010, and E168.

In some embodiments, ERBB2 is mutated to a point mutation in the amino acid sequence of ERBB 2. In some embodiments, the point mutation of ERBB2 is a point mutation that results in an amino acid substitution, results in mRNA splicing, or is a point mutation in the upstream region. Wherein the mutation comprises a nucleotide mutation that results in at least one amino acid substitution selected from the group consisting of Q568E, P601R, I628M, P885S, R143Q, R434Q and E874K.

In some embodiments, the ERBB2 mutation is an ERBB2 amplification. In some embodiments, ERBB2 amplification includes a point mutation selected from V659E, G309A, G309E, S310F, D769H, D769Y, V777L, P780ins, P780-Y781insGSP, V842I, R896C, K753E, and L755S, detectable by polymerase chain reaction or other sequencing techniques known in the art, e.g., as described in Bose et al, cancer discovery.2013, 3 (2), 224-237; and those of Zuo, et al clin Cancer res.2016,22 (19), 4859-4869, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, BRCA mutations are mutations in BRCA1 and/or BRCA2 (preferably BRCA 1) and/or in one or more other genes whose protein products are associated with BRCA1 and/or BRCA2 at the DNA damage site, including ATM, ATR, chk2, H2AX, 53BP1, NFBD1, mre11, rad50, nibrin, BRCA 1-related loop domain (BARD 1), abraxas, and MSH2. Mutations in more than one of these genes can result in gene expression patterns that mimic mutations in BRCA1 and/or BRCA 2.

In certain embodiments, the BRCA mutation comprises a non-synonymous mutation. In some embodiments, the BRCA mutation comprises a nonsensical mutation. In some embodiments, the BRCA mutation comprises an open reading frame shift mutation. In some embodiments, the BRCA mutation comprises a splice mutation. In some embodiments, BRCA is mutated to be expressed as mutant mRNA and final mutant protein. In some embodiments, the BRCA1/2 protein is functional. In other embodiments, the BRCA1/2 protein has reduced activity. In other embodiments, the BRCA1/2 protein is nonfunctional.

As used herein with respect to substitution, the "=" symbol with respect to mutation generally refers to synonymous substitution, silent codon, and/or silent substitution. In particular, synonymous substitution (also referred to as silent substitution or silent codon) refers to substitution of one nucleotide base in an exon of a gene encoding a protein for another, wherein the resulting amino acid sequence is unmodified. This is due to the fact that the genetic code is "degenerate," i.e., some amino acids are encoded by more than one three base pair codon. Because some codons for a given amino acid differ from other codons encoding the same amino acid by only one base pair, point mutations that replace the wild-type base by one alternative base will result in incorporation of the same amino acid into an elongated polypeptide chain during gene translation. In some embodiments, synonymous substitutions and mutations affecting non-coding DNA are generally considered silent mutations; however, these mutations are not always silent and have no effect. For example, synonymous mutations may affect transcription, splicing, mRNA transport, and translation, any of which may alter the resulting phenotype such that synonymous mutations are not silent. the substrate specificity of tRNA for rare codons can affect translation time, and thus co-translational folding of proteins. This is manifested in codon usage bias that has been observed in many species. Non-synonymous substitutions/mutations result in amino acid changes that can be arbitrarily categorized as conservative (change to an amino acid with similar physicochemical properties), semi-conservative (e.g., change from negatively charged to positively charged amino acid), or aggressive (very different amino acid). In some embodiments, BRCA mutations are BRCA1 mutations that include, but are not limited to, P871L, K1183R, D693N, S1634G, E1038G, S1040N, S694= (=). Silent codon), M1673I, Q356R, S1436=, L771=, K654 sfs=, L52C, E224C, E347=, S561C, E597, K820C, E893 Rfs 107, E962C, E1014C, E1258C, E1346 1347C, E1439 1472C, E1488, S1572 521602 1610C, E, Q1621=, Q5=, Q1625D 1626, and any combination thereof.

In some embodiments, BRCA mutations are BRCA2 mutations that include, but are not limited to, V2466A, N289H, N991D, S455= (=: silent codon), N372H, H743=, V1269=, S2414=, V2171=, L1521=, T3033Nfs = 11, K1132=, T3033Lfs 29, R2842C, N1784Tfs 7, K3326, D1420Y, I605Yfs x 9, I3412V, A2951T, T3085Nfs x 26, R2645Nfs x 3, S1013, T1915M, F3090=, V3244I, A1393, E2981R 37, N1784 x 3, K3416 x Nfs, K Nfs, K4891, S15, and any combination thereof.

In some embodiments, NRAS mutations include, but are not limited to, E63 61 61 12 13 61 61 12 12 12 12 61 13 61 12 61 13 13 13 13 13 61 61 12 60= (=: silence codons), Q61 61 146 59 59 61=, r68 146 12 62 75=, a91V, and any combination thereof.

E132K in some embodiments, PIK3CA mutations include substitution mutations, removal mutations, and insertion mutations. In some embodiments, the mutation occurs in the helical domain of PIK3CA and in its kinase. In other embodiments, in the P85BD domain of PIK3 CA. In some embodiments, the PIK3CA mutations are in exons 1, 2, 4, 5, 7, 9, 13, 18, and 20. In some embodiments, the PIK3CA mutation is in exons 9 and 20. In other embodiments, PIK3CA mutations are a combination of any of the above. Any combination of these exons may be tested, optionally in combination with testing other exons. The testing of mutations may be performed along the entire coding sequence or may be focused on the region where mutation aggregation has been found. Specific mutational hot spots occur at nucleotide positions 1624, 1633, 1636 and 3140 of the PIK3CA coding sequence.

In some embodiments, the size of the PIK3CA mutation is small, ranging from 1 to 3 nucleotides. In some embodiments, PIK3CA mutations include, but are not limited to, G1624A, G1633A, C1636A, A3140G, G113A, T1258C, G3129T, C3139T, E542K, E545K, Q R, H1047L, H1047R and G2702T.

In some embodiments, the MAP2K1 mutation is a MAP2K1 body mutation, optionally a MAP2K1 mutation that upregulates MEK1 levels. In some embodiments, the MAP2K1 mutation is a mutation in one or more genes associated with the RAS/MAPK pathway comprising: HRAS, KRAS, NRAS, ARAF, BRAF, RAFl, MAP2K2, MAPKl, MAPK3, MAP3K3. In certain embodiments, the MAP2K1 mutation is in more than one gene selected from RASA, PTEN, ENG, ACVRL1, SMAD4, GDF2, or a combination thereof.

In some embodiments, MAP2K1 mutations include, but are not limited to, P124S, Q P, K57N, E203K, G237, P124L, G128D, D67N, K57E, E102_i103del, C121S, K57T, K57N, Q P, P L, K N, G128V, Q58_e62del, f53L, I126=, i103_k104del, and any combination thereof.

In some embodiments, the KRAS mutation comprises a non-synonymous mutation. In some embodiments, the KRAS mutation comprises a nonsensical mutation. In some embodiments, the KRAS mutation comprises an open reading frame shift mutation. In some embodiments, the KRAS mutation comprises a splice mutation. In some embodiments, KRAS is mutated to be expressed as mutant mRNA and final mutant protein. In some embodiments, the mutated KRAS protein is functional. In other embodiments, the mutated KRAS protein has reduced activity. In other embodiments, the mutated KRAS protein is nonfunctional.

In some embodiments, KRAS mutations include, but are not limited to, G12 12 13 12 12 12 13 61 61 146 61 61 13 13 13 117 13 59 117 61 61 146 19 19 12 12 60=, G12=, G13=, a18 58 61 63 12 13 60 10dup, D57 59 14 33 12 13dup, and any combination thereof, wherein = indicates silence encoding.

In some embodiments, NF1 mutations include substitution mutations, deliberate mutations, missense mutations, aberrant splice mutations, and insertion mutations. In some embodiments, the NF1 mutation is a loss of function (LOF) mutation. In some embodiments, the NF1 mutation is selected from the group consisting of: R1947X (C5839T), R304X, exon 37 mutation, exon 4b mutation, exon 7 mutation, exon 10b mutation, and exon 10C mutation (e.g., 1570G→ T, E524X).

In some embodiments of the present invention, in some embodiments, the CDKN2A mutation includes, but is not limited to, R24P, D108G, D108N, D108Y, G125R, P114L, R, R58, H83Y, W110, P114L, E88, W110, E120, D108Y, D84Y, D, P81Y, D, L78Y, D, 41, D108Y, D, P48Y, D, Y44, E88Y, D80, D84Y, D16 Pfs, Y129, D108Y, D148Y, D36Y, D102Y, D15, H83Y, D57Y, D, D74Y, D153Y, D74Y, D5283Y, D82Y, D fs, Y129, E119, Y44, D74 5237_a19 dup, Y44L 32, L37del 28 del 33, D14D 16, D14L, or any combination thereof.

In some embodiments, the PTEN mutation comprises a non-synonymous mutation. In some embodiments, the PTEN mutation comprises a nonsensical mutation. In some embodiments, the PTEN mutation comprises an open reading frame shift mutation. In some embodiments, the PTEN mutation comprises a splice mutation. In some embodiments, the mutated PTEN is expressed as mRNA and final protein. In some embodiments, the mutated PTEN protein is functional. In other embodiments, the mutated PTEN protein has reduced activity. In other embodiments, the mutated PTEN protein is nonfunctional. In some embodiments, PTEN mutations include, but are not limited to, any of R130 319, R233, R130, K267Rfs 9, N323Mfs 21, N323 2, R173 335, Q171, Q245, E7, D268, 30, R130 214, R130 136 298, Q17, H93 Tfs 5, I33del, R233, E299, G132 68 319, N329, V166Sfs 14, V290, T319, T6, R142 38 126, 229, R130 129 Qfs 4, P246 130, G165 136 101 155 92, rfs 3, N184, G129E, R36 341, VG 123G 127 and 124, and combinations thereof.

In some embodiments, TP53 mutations include, but are not limited to, R175 245 248 248 249 273 273 273 282 141 151 158 173 173 173 176 179 179 179 220 237 238 245 245 249 273 278 280 285 158 195 214 266 266 266 278K or any combination thereof. In some further embodiments, the TP53 mutation is selected from the group consisting of: G245S, R249S, R273C, R273H, C141Y, V157F, R158L, Y163C, V173L, V173M, Y205C, Y220C, G245C, R249M, V272M, R L and E286K. In some embodiments, the TP53 mutation comprises one or more of the above mutations.

In some embodiments, CREBBP mutations include, but are not limited to, R1446C, R1446H, S1680del, I1084Sfs 15, P1948L, I1084Nfs x 3,? R386, S893L, R1341, P1423Lfs 36, P1488L, Y1503H, R1664C, A1824C, A1173, R1360, Y1450C, A2228C, A928 =, D1435C, A1502C, A1503C, A483, R601C, A945C, A1103, R1288C, A1392, C1408C, A1446C, A1485C, A1491C, A96L 361C, A Wfs 6, Q540, Q1073, a 1100C, A1169C, A1237C, A1347C, A1411C, A1472C, A1488C, A1498, Y1503C, A1856, R1985C, A2104C, A2328C, A2349=, S2377L, and any combination thereof.

In some embodiments, KMT2C mutations include, but are not limited to, D348N, P350 =, R380L, C391 =, P309S, C988F, Y987H, S990G, K Rfs =, V346 =, R894Q, R284Q, S =, R1690 =, P986 =, a1685S, G315S, Q =, R909K, T316S, S772L, G838S, L291F, P335 =, C988F, Q2680 =, E765G, K339N, Y816, R526P, N729D, G845E, I817Nfs =, G892R, C =, S3660L, F Lfs 315 =, G C, R =, G48886C, D =, V919C, D =, P9837C, D =, R C, D D37 =, T C, D =, T37 =, D37 =, T37 =.

In some embodiments, KMT2D mutations include, but are not limited to, L1419P, E640D, E541D, E455D, T2131P, K1420R, P2354Lfs 30, G2493=, Q3612=, I942=, T1195Hfs =, P4170=, P1194H, G1235Vfs 95, P4563=, P647Hfs =, L449_p457del, P3557=, Q3603=, R1702, P648Tfs 2, R5501, R4198, R4484, R83Q, R1903, R2685, R4282, L5326 =, R5432W, R2734, Q2800, R2830, Q3745dup, S4010P, R4904, G5182Afs x 61, R5214H, R1615, Q2380, R2687, R2771, V3089Wfs 30, Q3799Gfs x 212, R4536 x, R5030C, R5048C, R5432Q, A Lfs 40, a476T, A lx9 Lfs 25, P2557l, R2801, Q3913, R4420W, G4641 =, R5097 x, and any combination thereof.

In some embodiments, ARID1A mutations include, but are not limited to, for example, mutations of ARID1A in a subject selected from C884 x (nonsensical mutation), E966K, Q1411 x, F1720fs (fs: frame shift), G1847fs, C1874fs, D1957E, Q1430, R1721fs, G1255E, G284fs, R1722 x, M274fs, G1847fs, P559fs, R1276 x, Q2176fs, H203fs, a591fs, Q1322 x, S2264 x, Q586 x, Q548fs, and N756 fs.

In some embodiments, RB1 mutations include, but are not limited to, R320X, R467X, R579X, R455 3835X, R X, R787X, R552X, R X, R556X, Y X, Q575X, E323X, R661W, R579, R455, R556, R787, R661 35445, R467, Q217, Q471, W195, Q395, I680 37137, R255, Q344, Q62, E440K, A488V, P777Lfs 33, E322K, R656W, G Rfs 36, C221, E440, Q93, Q504, E125, S834, E323, Q685, S829, W516, G435, Q257, E5679, E567, S6767, S6314, S11, S654 and any combination thereof.

In some embodiments, ATM mutations are mutations in the ATM gene sequence, including, but not limited to 10744a > g;10744a > g;11482g > a; IVS3-558A > T;146C > G;381delA; IVS8-3delGT;1028delAAAA;1120C > T;1930ins16; IVS16+2T > C;2572t > c; IVS21+1G > A;3085delA;3381delTGAC;3602delTT;4052delT; 43966C > T;5188C > T;5290delC;5546delT;5791g > cct;6047a > g; IVS44-1G > T;6672delGC/6677delTACG;6736dell 1/6749del7;7159insAGCC;7671delGTTT;7705del14;7865c > t;7979delTGT;8177c > t;8545C > T;8565T > A; IVS64+1G > T; and 9010del28.

In some embodiments of the invention, when the transcription initiation codon position of the mRNA sequence of NCBI accession No. nm_014159 is set to 1, SETD2 is mutated to an alteration in the gene sequence encoding SETD2 protein. In some embodiments, position G (guanine) is substituted with T (thymine), position C (cytosine) is substituted with T, position 1210 is substituted with T, position a (adenine) is substituted with G, position 5290 is substituted with T, position C is substituted with T, position 7072 is substituted with T, position G is substituted with T, position 1297 is substituted with T, position T is substituted with G, position 7261 is substituted with G, position 6700 is substituted with T, position 2536 is substituted with T, position C is substituted with T or a is inserted at position 3866, T is inserted at position 6712, T is inserted at position 7572, position a is divided by position 913, position C is divided by 5619, bases 4603-4604 are divided by 1, base is divided by position C is divided by 1936, base is divided by 3094-3118, and bases are inserted at positions 5289 and 6323-6333.

In some embodiments, FLT3 mutations include, but are not limited to, (q569_e648) ins, D835X, (q569_e648) delins, (D835_i 836), D835 835 835 835 835 227 836del, N676 835 597_e598 835del, f594_d600dup, a680 839 96=, D835 491 835 989, D835 561=, I836del, P986X 27, D7 324 451 835 576 597_e598 insdfrey, V491 841 324 595_l601dup, K663 67691 835 836 993 832V, and any combination thereof.

In some embodiments, PTPN11 mutations include, but are not limited to, 76 72 72 61 60 69 76 507 73 76 76 61 71 76 71 72 72 472 495 58 285 189 465 507 511 61 61 60 60 514 197, N308 514 58 206=, a465 495 507R, and any combination thereof.

In some embodiments, FGFR1 mutations include, but are not limited to, N577K, K687E, N577K, D del, T371M, R476W, T350=, E498K, N D, D683G, R87C, A154D, N =, a374V, D =, S633=, V695L, G728=, R765W, P S, W19C, P =, R113C, V149 42158L, D166dupR220C, N224C, N x 8, D249C, N281C, N299C, N424C, N461C, N4637 52506Q, and any combination thereof.

In some embodiments, EP300 mutations include, but are not limited to, D1399N, Y1414, cfs 26, Y1111, H2324Pfs 55, R1627N, Y2209_q 2213 delnsk, Q2268del, L415N, Y1470, N, Y3, E1514N, Y1201 1452, C1164N, Y1399, N, Y, 507, D1507N, Y2324 Tfs 29, P925N, Y, N, Y1629, 1645, N1700Tfs 9, P1869N, Y65, a 171N, Y, R580N, Y, N, Y1082, N1236N, Y, N1286N, Y, R1386, C5N, Y, N, Y1N, Y2, Y1467N, Y, R14637, and any combination thereof.

In some embodiments of the present invention, in some embodiments, MYC mutations include, but are not limited to, 41 amino acid solutions at end E61 68 74 75 135 394 420 96 325, deleted MYC protein (dN 2 MYC), N26 161 74 7 153 54 246, L164 74 59 73 72 73 374 73 264 72 52del, S21 74 107 75 77 261 74 73 11 21 78 9 190 267 73 105 187 71 10 191x, Q50x, L191 25 130 27 195 20 6 20 2 75 152 40 8 48x, and any combination thereof.

In some embodiments, the EZH2 mutation is associated with an altered histone methylation pattern. In some embodiments, the EZH2 mutation results in the conversion of amino acid Y641 (corresponding to catalytic domain Y646) to F, N, H, S or C, resulting in Gao Sanjia glycosylation of H3K27 and driving lymphogenesis. In some embodiments, the EZH2 mutations include EZH2SET domain mutations, overexpression of EZH2, overexpression of other PRC2 subunits, loss of function mutations of Histone Acetyl Transferase (HAT), and loss of function of MLL 2. Cells of the EZH2Y646 mutant homozygote resulted in Gao Sanjia glycosylation of H3K27 relative to cells of the EZH2 protein wild-type (WT) homozygote or cells of the Y646 mutant homozygote.

In some embodiments of the present invention, in some embodiments, EZH2 mutations include, but are not limited to Y646 646 185 646 646 646 646 646 646 646 646 646 646 646 626 679 690 684 682 249 159 288 322 692 690 x (insertion reading frame shift) S695 684 667 288, S644, D192 550 653 664 646 660 213 255 538 693 55 561 692 515 733R 63, Q570, Q328, R25 467 656 573 571 16 577 145 680 686 145 298 566 149, 731 675, 652 374, N152Ifs 15, E401 x 22, K406Mfs 17, E246 x, S624 146 626 674 694 581S, and any combination thereof.

In some embodiments, the mutation of JAK2 in the JAK2 gene includes, but is not limited to, a combination of a T1923C mutation with a G1920T mutation, a G1920T/C1922T mutation, or a G1920A mutation. In some embodiments, JAK2 mutations are mutant JAK2 proteins comprising more than one substitution including, but not limited to, V617 617 617 683 542_e543del, E543_d544del, R683 683 537_k539 delnsl (in frame, divided), K539 1108 1113 1063 487 Mfs 3 (in frame shift removed), R867 539 571 1113 938 228, E1080, K539 618 564 1036 1088 538, D873 682 393 535 875 921 611 87611 921 538 1035L, and any combination thereof.

In some embodiments, the FBXW7 mutation is a point mutation selected from the group consisting of W244 (: stop codon), R222, R278, E192A, S, E113D, R H/C, 726+1g > a splicing, R505C, R479Q, R465C, R367, R499Vfs 25 (fs: frame shift), R658, D600Y, D35520N, D Y, and any combination thereof. In further embodiments, the FBXW7 mutation is a double mutation or a triple mutation, including but not limited to R479Q and S582L, R465H and S582L, D35520N, D Y and R14Q and R367 x and S582L.

In some embodiments, CCND3 mutations include, but are not limited to, S259A, R271Pfs 53 (insertion causes frame shift), E51, Q260, P199S, T283A, T283P, V287D, D286_t288del, R271Gfs x 33, Q276, R241Q, D238G, R33P, I290K, I290T, I290R, P267fs, P284S, P284L, P100S, E253D, S262I, R W, R114L, D238N, A266E, R W, and any combination thereof.

In some embodiments, GNA11 mutations include, but are not limited to, Q209L, R183C, T257 =, R183C, G208Afs x 16, Q209H, R183C, Q209P, Q209R, Q H,? T96=, R210W, R256Q, T334=, G48D, S53G, Q209P, R Q, and any combination thereof. In some embodiments, the GNA11 mutation has two mutations in exon 4, e.g., a mutation in V182 and a mutation in T175 or more than one mutation in exon 5.

2. In combination with PD-1 and PD-L1 inhibitors

In some embodiments, the TIL therapy provided to a cancer patient may include a monotherapy TIL population therapy or may include a combination therapy including 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 CD 279) belongs to the CD28 family, and is encoded in humans by the Pdcd1 gene on chromosome 2. PD-1 consists of an immunoglobulin (Ig) superfamily domain, a transmembrane region, and an intracellular domain containing an immunoreceptor tyrosine basal-inhibiting motif (ITIM) and an immunoreceptor tyrosine basal-switching motif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play a key role in immune tolerance as described by Keir et al, annu.Rev.Immunol.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 can 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 with PD-1 inhibitors, PD-L1 inhibitors and/or PD-L2 inhibitors may overcome immune resistance as shown by recent clinical studies such as Topalian et al, n.eng.j. Med.2012,366, 2443-54. PD-L1 is expressed on many tumor cell lines, and PD-L2 is expressed mostly on dendritic cells and a few tumor cell lines. 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 one embodiment, 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 detailed in the following paragraphs. The terms "inhibitor", "antagonist" and "blocker" are used interchangeably herein with reference to PD-1 inhibitors. 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, reference 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 a preferred embodiment, the PD-1 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof (including Fab fragments), or a single chain variable fragment (scFv) thereof. In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In a preferred embodiment, 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 one embodiment, the antibody competes for binding with PD-1 and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is at a K of about 100pM or less _D Binds to human PD-1 with a K of about 90pM or less _D Binds to human PD-1 with a K of about 80pM or less _D Binds to human PD-1 with a K of about 70pM or less _D Binds to human PD-1 with a K of about 60pM or less _D Binds to human PD-1 with a K of about 50pM or less _D Binds to human PD-1 with a K of about 40pM or less _D Binds to human PD-1 with a K of about 30pM or less _D Binds to human PD-1 with a K of about 20pM or less _D Binds to human PD-1 with a K of about 10pM or less _D Binds to human PD-1 or at a K of about 1pM or less _D PD-1 inhibitors that bind to human PD-1.

In some embodiments, the PD-1 inhibitor is present at about 7.5 x 10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-1 at about 7.5X10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-1 at about 8X 10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-1 at about 8.5 x 10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-1 at about 9X 10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-1 at about 9.5 x 10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-1 or at about 1X 10 ⁶ K of 1/M.s or more _{Association with} PD-1 inhibitors that bind to human PD-1.

In some embodiments, the PD-1 inhibitor is present in an amount of about 2 x 10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.1X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.2 x 10 ^-5 K of 1/s or less _{Dissociation of} And human PD-1Combined, at about 2.3X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.4X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.5X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.6X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 or at about 2.7X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.8x10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.9x10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 or at about 3X 10 ^-5 K of 1/s or less _{Dissociation of} PD-1 inhibitors that bind to human PD-1.

In some embodiments, the PD-1 inhibitor is an IC of about 10nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 9nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 8nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 7nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 6nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 5nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 4nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 3nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 with an IC of about 2nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1 or IC at about 1nM or less ₅₀ PD-1 inhibitors that block or inhibit binding of human PD-L1 or human PD-L2 to human PD-1.

In one embodiment, the PD-1 inhibitor is nivolumab (Bristol-Myers Squibb co. Commercially available as 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 one embodiment, the anti-PD-1 antibody is an immunoglobulin G4 k, anti- (human CD 274) antibody. Nivolumab was assigned Chemical Abstracts (CAS) accession numbers 946414-94-4, also known as 5C4, BMS-936558, MDX-1106, and ONO-4538. The preparation and properties of nivolumab are described in U.S. patent No. 8,008,449 and international patent publication WO 2006/121168, the disclosures of which are incorporated herein by reference in their entirety. Clinical safety and efficacy of nivolumab in various forms of Cancer has been described in Wang et al, cancer immunol.res.2014,2,846-56; page et al, ann.Rev.Med.,2014,65,185-202; and Weber et al, j.clin.oncology,2013,31,4311-4318, the disclosures of which are incorporated herein by reference in their entirety. The amino acid sequence of nivolumab is shown in table 18. Nivolumab has disulfide bonds within the heavy chains located at 22-96, 140-196, 254-314, 360-418, 22"-96", 140"-196", 254"-314" and 360 "-418"; disulfide bonds within the light chains of 23'-88', 134'-194', 23 '"-88'" and 134 '"-194'"; heavy-light interchain disulfide bonds at 127-214', 127 "-214'", heavy-heavy interchain disulfide bonds at 219-219 "and 222-222"; and an N-glycosylation site at 290, 290' (HCH 2 84.4).

In one embodiment, the PD-1 inhibitor comprises an amino acid sequence consisting of SEQ ID NO:158 and the heavy chain represented by SEQ ID NO: 159. In one embodiment, 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 one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 99% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 98% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 97% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:158 and SEQ ID NO:159, and a heavy chain and a light chain having at least 96% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:463 and SEQ ID NO:159, and a heavy chain and a light chain having at least 95% identity.

In one embodiment, the PD-1 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of nivolumab. In one embodiment, the PD-1 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:160, and a PD-1 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:161 or a conservative amino acid substitution thereof. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:160 and SEQ ID NO:161 has at least 99% identity V _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:160 and SEQ ID NO:161 has a V with at least 98% identity to the sequence shown in 161 _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:160 and SEQ ID NO:161 has at least 97% identity V _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:160 and SEQ ID NO:161 has a V with at least 96% identity to the sequence shown in 161 _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:160 and SEQ ID NO:161 has a V with at least 95% identity to the sequence shown in 161 _H And V _L A zone.

In one embodiment, the PD-1 inhibitor comprises a polypeptide having the sequence set forth in SEQ ID NO: 162. SEQ ID NO:163 and SEQ ID NO:164 or conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 165. SEQ ID NO:166 and SEQ ID NO:167 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown in fig. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on PD-1 and/or bind to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the PD-1 inhibitor is an anti-PD-1 biological analog monoclonal antibody approved by the drug administration with reference to nivolumab. In one embodiment, the 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 the 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, which 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, and the anti-PD-1 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is nivolumab. anti-PD-1 antibodies may be licensed by pharmaceutical authorities such as the FDA in the united states and/or EMA in the european union. In some embodiments, the biological analog is a composition provided further comprising one or more excipients, the one or more excipients being the same as or different from excipients included in a reference drug or reference biological product, the reference drug or reference biological product being nivolumab. In some embodiments, the biological analog is a composition provided further comprising one or more excipients, the one or more excipients being the same as or different from excipients included in a reference drug or reference biological product, the reference drug or reference biological product being nivolumab.

Table 18: amino acid sequence of PD-1 inhibitor related to nivolumab

In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, and nivolumab is administered at a dose of about 0.5mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, which is administered at a dose of 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 10 mg/kg. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 nivolumab is administered at a dose of about 200mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biological analog thereof, which is administered at a dose of 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 500 mg. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 nivolumab is administered once every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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, nivolumab is administered to treat unresectable or metastatic melanoma and at about 240mg every 2 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma and at about 480mg every 4 weeks. In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma at about 1mg/kg every 3 weeks followed by 4 doses of 3mg/kg of ipilimab on the same day, followed by 240mg every 2 weeks or 480mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 as an adjuvant treatment for melanoma. In some embodiments, nivolumab is administered at about 240mg every 2 weeks for adjuvant treatment of melanoma. In some embodiments, nivolumab is administered at about 480mg every 4 weeks for adjuvant treatment of melanoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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, along with about 1mg/kg of ipilimab every 6 weeks to treat metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered at about 360mg every 3 weeks, along with 1mg/kg of ipilimumab every 6 weeks and 2 cycles of platinum 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, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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, nivolumab is administered at about 240mg every 2 weeks to treat small cell lung cancer. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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, along with 1mg/kg of ipilimumab every 6 weeks to treat malignant pleural mesothelioma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 every 3 weeks followed by a total of 4 doses of about 1mg/kg of ipilimumab on the same day, followed by 240mg every 2 weeks to treat advanced renal cell carcinoma. In some embodiments, nivolumab is administered at about 3mg/kg every 3 weeks followed by a total of 4 doses of about 1mg/kg of ipilimumab on the same day, followed by 240mg every 2 weeks and 480mg every 4 weeks to treat advanced renal cell carcinoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab may 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 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, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 urinary epithelial cancer. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat locally advanced or metastatic urinary epithelial cancer. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer. In some embodiments, nivolumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in an adult or pediatric patient. In some embodiments, nivolumab is administered at about 240mg every 2 weeks to treat ≡40kg of microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult or pediatric patients. In some embodiments, nivolumab is administered at about 480mg every 4 weeks to treat ≡40kg of microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult or pediatric patients. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 microsatellite high 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 every 3 weeks followed by a total of 4 doses of 1mg/kg of ipilimab on the same day, followed by 240mg every 2 weeks to treat ≡40kg of microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult or pediatric patients. In some embodiments, nivolumab is administered at about 3mg/kg every 3 weeks followed by 4 doses of 1mg/kg of ipilimab on the same day, followed by 480mg every 4 weeks to treat ≡40kg of microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer in adult or pediatric patients. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 every 3 weeks followed by 4 doses of 3mg/kg of ipilimab on the same day, followed by 240mg every 2 weeks to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered at about 1mg/kg every 3 weeks followed by 4 doses of 3mg/kg of ipilimab on the same day, followed by 480mg every 4 weeks to treat hepatocellular carcinoma. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. 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 another embodiment, the PD-1 inhibitor comprises pembrolizumab (Merck & co., inc., kenilworth, NJ, USA, commercially available as keyruda) or an antigen-binding fragment, conjugate, or variant thereof. Pemumab is assigned CAS registry number 1374853-91-4, also known as lebelide-I-MAB (lambrolizumab), MK-3475 and SCH-900475. Pembrolizumab has a dimeric structure of immunoglobulin G4, anti- (human protein PDCD1 (programmed cell death 1)) (human-mouse monoclonal heavy chain) and human-mouse monoclonal light chain disulfide. The structure of pembrolizumab can also be described as immunoglobulin G4, anti- (human programmed cell death 1); humanized mouse monoclonal [ 228-L-proline (H10-S > P) ] gamma 4 heavy chain (134-218') -disulfide and humanized mouse monoclonal kappa light chain dimer (226-226 ": 229-229") -double disulfide. The nature, use and preparation of pembrolizumab are described in international patent publication No. WO 2008/156712 A1, U.S. patent No. 8,354,509, U.S. patent application publication nos. US 2010/0266617 A1, US 2013/0108651 A1 and US 2013/0109843 A2, the disclosures of which are incorporated herein by reference in their entirety. The clinical safety and efficacy of pembrolizumab in various forms of cancer has been described in furst, oncology Times,2014,36,35-36; robert et al, lancet,2014,384,1109-17; and Thomas et al, exp.opin.biol.ter., 2014,14,1061-1064. The amino acid sequence of pembrolizumab is shown in table 19. Pembrolizumab includes 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" and the following glycosylation sites (N): asn-297 and Asn-297). Pembrolizumab is of IgG4/κ isotype, with a stable S228P mutation in the Fc region; insertion of this mutation in the IgG4 hinge region prevents the formation of half-molecules typically observed in IgG4 antibodies. Pembrolizumab is heterogeneously glycosylated at Asn297 within each heavy chain Fc domain to yield a molecular weight of about 149kDa for the intact antibody. The dominant glycosylated form of pembrolizumab is the fucosylated form of galactose-free biantennary glycan (G0F).

In one embodiment, the PD-1 inhibitor comprises an amino acid sequence consisting of SEQ ID NO:168 and the heavy chain given by SEQ ID NO: 169. In one embodiment, 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 one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:168 and SEQ ID NO:169 have at least 99% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:168 and SEQ ID NO:169 have a heavy chain and a light chain having at least 98% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:168 and SEQ ID NO:169 have a heavy chain and a light chain having at least 97% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:168 and SEQ ID NO:169 have a heavy chain and a light chain having at least 96% identity. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:168 and SEQ ID NO:169 have at least 95% identity.

In one embodiment, the PD-1 inhibitor comprises heavy and light chain CDRs or Variable Regions (VR) of pembrolizumab. In one embodiment, the PD-1 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:170, and a PD-1 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:171 or conservative amino acid substitutions thereof. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:170 and SEQ ID NO:171 has at least 99% identity V _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:170 and SEQ ID NO:171 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:170 and SEQ ID NO:171 has at least 97% identity V _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:170 and SEQ ID NO:171 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the PD-1 inhibitor comprises an amino acid sequence that is each of SEQ ID NO:170 and SEQ ID NO:171 has at least 95% identity of V _H And V _L A zone.

In one embodiment, the PD-1 inhibitor comprises a polypeptide having the sequence set forth in SEQ ID NO: 172. SEQ ID NO:173 and SEQ ID NO:174 or conservative amino acid substitutions thereof, and having heavy chain CDR1, CDR2 and CDR3 domains of the sequences set forth in SEQ ID NO: 175. SEQ ID NO:176 and SEQ ID NO:177, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on PD-1 and/or bind to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the PD-1 inhibitor is an anti-PD-1 biological analog monoclonal antibody approved by a drug administration reference pembrolizumab. In one embodiment, the 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 the 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, which is pembrolizumab. 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, and the anti-PD-1 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is pembrolizumab. anti-PD-1 antibodies may be licensed by pharmaceutical authorities such as the FDA in the united states and/or EMA in the european union. In some embodiments, the biological analog 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 biological product, which is pembrolizumab. In some embodiments, the biological analog 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 biological product, which is pembrolizumab.

Table 19: amino acid sequence of PD-1 inhibitor related to pembrolizumab

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biological analog thereof, and pembrolizumab is administered at a dose of about 0.5mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biological analog thereof, and pembrolizumab is administered at a dose of 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 10 mg/kg. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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 pembrolizumab or a biological analog thereof, wherein pembrolizumab is administered at a dose of about 200mg to about 500 mg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biological analog thereof, and nivolumab is administered at a dose of 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 500 mg. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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 pembrolizumab or a biological analog thereof, wherein pembrolizumab is administered once every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered to treat melanoma. In some embodiments, pembrolizumab is administered at about 200mg every 3 weeks for the treatment of melanoma. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks for the treatment of melanoma. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered to treat NSCLC. In some embodiments, pembrolizumab is administered at about 200mg every 3 weeks to treat NSCLC. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat NSCLC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered to treat Small Cell Lung Cancer (SCLC). In some embodiments, pembrolizumab is administered at about 200mg every 3 weeks for the treatment of SCLC. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks for the treatment of SCLC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered to treat Head and Neck Squamous Cell Carcinoma (HNSCC). In some embodiments, pembrolizumab is administered at about 200mg every 3 weeks to treat HNSCC. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat HNSCC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat a typical hodgkin's lymphoma (cHL) or primary mediastinal large B cell lymphoma (PMBCL). In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat typical hodgkin's lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL) in adults. In some embodiments, pembrolizumab is administered at about 2mg/kg (up to 200 mg) every 3 weeks to treat a typical hodgkin's lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL) in a child. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat urothelial cancer. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat urothelial cancer. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) cancer. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat MSI-H or dhmr cancer in adults. In some embodiments, pembrolizumab is administered at about 2mg/kg (up to 200 mg) every 3 weeks to treat pediatric MSI-H or dhmr cancers. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat microsatellite high instability (MSI-H) or mismatch repair deficient colorectal cancer (dMMR CRC). In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat MSI-H or dhmr CRC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat gastric cancer. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat gastric cancer. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat esophageal cancer. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat esophageal cancer. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks for the treatment of cervical cancer. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks for the treatment of cervical cancer. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat hepatocellular carcinoma (HCC). In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks for the treatment of HCC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat Merkel's Cell Carcinoma (MCC) in adults. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat adult MCC. In some embodiments, pembrolizumab is administered at about 2mg/kg (up to 200 mg) every 3 weeks to treat pediatric MCC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat Renal Cell Carcinoma (RCC). In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks, along with 5mg of twice daily oral axitinib (axitinib) to treat RCC in adults. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks for the treatment of endometrial cancer. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks in conjunction with 20mg of once daily oral lenvatinib (lenvatinib) to treat tumor endometrial cancer that is not MSI-H or dMMR. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat high tumor mutational burden (TMB-H) cancer in adults. In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat TMB-H cancer in adults. In some embodiments, pembrolizumab is administered at about 2mg/kg (up to 200 mg) every 3 weeks to treat pediatric TMB-H cancer. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat cutaneous squamous cell carcinoma (cSCC). In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks to treat cSCC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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, pembrolizumab is administered at about 200mg every 3 weeks to treat Triple Negative Breast Cancer (TNBC). In some embodiments, pembrolizumab is administered at about 400mg every 6 weeks for treating TNBC. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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 one embodiment, if the patient or subject is an adult (i.e., treatment of adult indications), an additional dosing regimen of 400mg every 6 weeks may be employed. In some embodiments, pembrolii Shan Kangshi begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolii Shan Kangshi begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab can 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, pembrolizumab can 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 one embodiment, the PD-1 inhibitor or anti-PD-1 antibody is sibirizumab or a fragment, variant, conjugate, or biological analog thereof, commercially available from Regeneron, inc. In one embodiment, the PD-1 inhibitor or anti-PD-1 antibody is tirelimumab or a fragment, variant, conjugate, or biological analog thereof, available from Novartis AG and Beigene co. In one embodiment, the PD-1 inhibitor or anti-PD-1 antibody is a signal di Li Shan antibody or fragment, variant, conjugate or biological analog thereof, available from Eli Lilly and co. In one embodiment, the PD-1 inhibitor or anti-PD-1 antibody is terlipressin Li Shan antibody or a fragment, variant, conjugate or biological analog thereof, available from Junshi Biosciences co., ltd. In one embodiment, the PD-1 inhibitor or anti-PD-1 antibody is a doslimab or fragment, variant, conjugate, or biological analog thereof, available from GlaxoSmithKline plc.

In one embodiment, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 clone J43 (Cat#BE 0033-2) and RMP1-14 (Cat#BE 0146) (Bio X Cell, inc., celebaner, new Hampshish, U.S.A.). Some commercially available anti-PD-1 antibodies are known to those skilled in the art.

In one embodiment, the PD-1 inhibitor is an antibody disclosed in U.S. patent No. 8,354,509 or U.S. patent application publication nos. 2010/0266617 A1, 2013/0108651 A1, 2013/0109843 A2, the disclosures of which are incorporated herein by reference in their entirety. In one embodiment, PD-1 inhibitors are described in U.S. patent nos. 8,287,856, 8,580,247, and 8,168,757, and U.S. patent application publication nos. 2009/0028857 A1, 2010/0285013 A1, 2013/0022600 A1, and 2011/0008369 A1 (the teachings of which are incorporated herein by reference in their entirety). In another embodiment, 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 its entirety. In one embodiment, the PD-1 inhibitor is pilizumab (also known as CT-011), which is described in U.S. patent No. 8,686,119, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the PD-1 inhibitor may be a small molecule or a peptide or peptide derivative (e.g., those described in U.S. Pat. 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 (e.g., those described in U.S. patent application publication No. 2015/0074302); cyclic peptidomimetic compounds and derivatives (e.g., those described in U.S. patent application publication No. US 2015/007402); 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 (e.g., those 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 macrocyclic peptide-based compounds and derivatives (such as those described in U.S. patent application publication No. US 2014/0294898); the respective disclosures of which are incorporated herein by reference in their entirety.

In one embodiment, 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 detailed in the following paragraphs. The terms "inhibitor", "antagonist" and "blocker" are used interchangeably herein with reference to PD-L1 and PD-L2 inhibitors. 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 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 a preferred embodiment, the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti-PD-1 antibody), a fragment thereof (including Fab fragments), or a single chain variable fragment (scFv) thereof. In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In a preferred embodiment, 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 binds to an epitope on PD-L1 or PD-L2. In one embodiment, 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, i.e., the compound binds to or interacts with PD-L1 at a concentration substantially lower than it binds to 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 to the PD-L2 receptor.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2, i.e., the compound binds to or interacts with PD-L2 at a concentration substantially lower than it binds to 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 to 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, PD-L1 expression by tumor cells is not necessary for the efficacy of a PD-1 inhibitor or blocker or PD-L1 inhibitor or blocker. In one embodiment, the tumor cell expresses PD-L1. In another embodiment, 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., those described herein) in combination with TIL. The combination of PD-1 and PD-L1 antibodies and TIL may be administered simultaneously or sequentially.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is at a K of about 100pM or less _D Binds to human PD-L1 and/or PD-L2 at a K of about 90pM or less _D Binds to human PD-L1 and/or PD-L2 at a K of about 80pM or less _D Binds to human PD-L1 and/or PD-L2 at a K of about 70pM or less _D Binds to human PD-L1 and/or PD-L2 at a K of about 60pM or less _D At a K of about 50pM or less _D Binds to human PD-L1 and/or PD-L2 at a K of about 40pM or less _D Binds to human PD-L1 and/or PD-L2 or has a K of about 30pM or less _D PD-L1 and/or PD-L2 inhibitors that bind to human PD-L1 and/or PD-L2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is at about 7.5 x 10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-L1 and/or PD-L2 at about 8X10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-L1 and/or PD-L2 at about 8.5X10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-L1 and/or PD-L2 at about 9X 10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-L1 and/or PD-L2 at about 9.5X10 ⁵ K of 1/M.s or more _{Association with} Binds to human PD-L1 and/or PD-L2 or at about 1X 10 ⁶ K of 1/M.s or more _{Association with} PD-L1 and/or PD-L2 inhibitors that bind to human PD-L1 and/or PD-L2.

In some implementationsIn embodiments, the PD-L1 and/or PD-L2 inhibitor is at a k of about 2X 10-5 1/s or less _{Dissociation of} Binds to human PD-L1 or PD-L2 at about 2.1X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.2 x 10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.3 x 10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.4X10 ^- ⁵ K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.5X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.6X10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-1 at about 2.7x10 ^-5 K of 1/s or less _{Dissociation of} Binds to human PD-L1 or PD-L2 or at about 3X 10 ^-5 K of 1/s or less _{Dissociation of} PD-L1 and/or PD-L2 inhibitors that bind to human PD-L1 or PD-L2.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is an IC of about 10nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; with an IC of about 9nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; IC at about 8nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; IC at about 7nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; with an IC of about 6nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; with an IC of about 5nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; IC of about 4nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; with an IC of about 3nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; IC of about 2nM or less ₅₀ Blocking or inhibiting binding of human PD-L1 or human PD-L2 to human PD-1; or IC of about 1nM or less ₅₀ PD-L1 and/or PD-L2 inhibitors that block human PD-1 or block human PD-L1 or human PD-L2 from binding to human PD-1.

In one embodiment, the PD-L1 inhibitor is dewaruzumab, also known as MEDI4736 (a subsidiary company of AstraZeneca plc, LLC, gaithersburg, maryland) or an antigen-binding fragment, conjugate, or variant thereof. In one embodiment, 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 in their entirety. The clinical efficacy of Dewaruzumab has been described in Page et al, ann.Rev.Med.,2014,65,185-202; brahmer et al, j.clin.oncol.2014,32,5s (support, abscist 8021); and McDermott et al, cancer Treatment Rev.,2014,40,1056-64. The preparation and properties of Dewaruzumab are described in U.S. Pat. No. 8,779,108, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of Dewaruzumab are shown in Table 20. 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 located at Asn-301 and Asn-301'.

In one embodiment, the PD-L1 inhibitor comprises SEQ ID NO:178 and SEQ ID NO: 179. In one embodiment, 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 one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 99% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 98% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 97% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 96% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:178 and SEQ ID NO:179 have a heavy chain and a light chain with at least 95% identity.

In one embodiment, the PD-L1 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of dewarfarin. In one embodiment, the PD-L1 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:180, a PD-L1 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:181 or conservative amino acid substitutions thereof. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:180 and SEQ ID NO:181 has at least 99% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:180 and SEQ ID NO:181 has at least 98% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:180 and SEQ ID NO:181 has at least 97% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:180 and SEQ ID NO:181 has at least 96% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:180 and SEQ ID NO:181 has at least 95% identity V _H And V _L A zone.

In one embodiment, the PD-L1 inhibitor comprises a polypeptide having the sequence set forth in SEQ ID NO: 182. SEQ ID NO:183 and SEQ ID NO:184 or conservative amino acid substitutions thereof, and having heavy chain CDR1, CDR2 and CDR3 domains of the sequences set forth in SEQ ID NOs: 185. SEQ ID NO:186 and SEQ ID NO:187, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on PD-L1 and/or bind to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 biological analog monoclonal antibody approved by the drug administration with reference to de valuzumab. In one embodiment, the 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 the 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, which is de valuzumab. 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, and the anti-PD-L1 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is de-valuzumab. anti-PD-L1 antibodies may be licensed by drug administration such as the us FDA and/or EMA of the european union. In some embodiments, the biosimilar is a composition provided further comprising one or more excipients that are the same as or different from excipients included in a reference drug or reference biologic, which is Devaluzumab. In some embodiments, the biosimilar is a composition provided further comprising one or more excipients that are the same as or different from excipients included in a reference drug or reference biologic, which is Devaluzumab.

Table 20: amino acid sequence of PD-L1 inhibitor related to Dewaruzumab

In one embodiment, the PD-L1 inhibitor is esvaluzumab, also known as MSB0010718C (commercially available from Merck KGaA/EMD Serono) or an antigen-binding fragment, conjugate, or variant thereof. The preparation and properties of esvaluzumab are described in U.S. patent application publication No. US 2014/0341917 A1, the disclosure of which is specifically incorporated herein by reference in its entirety. The amino acid sequence of esvaluzumab is shown in table 21. Evellumab has disulfide bonds (C23-C104) within the heavy chains of 22-96, 147-203, 264-324, 370-428, 22"-96", 147"-203", 264"-324" and 370 "-428"; at 22'-90', 138 '-197': 22 '"-90'" and 138 '"-197'" are disulfide bonds (C23-C104); disulfide bonds within the heavy-light chains located at 223-215 'and 223 "-215'" (h 5-CL 126); disulfide bonds (h 11, h 14) within the heavy chain-heavy chain located at 229-229 "and 232-232"; n-glycosylation sites at 300, 300' (H CH2N84.4); fucosylation complex double antennary CHO-type glycans; and H CHS K2C-terminal lysine truncations at 450 and 450'.

In one embodiment, the PD-L1 inhibitor comprises SEQ ID NO:188 and SEQ ID NO: 189. In one embodiment, 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 one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:188 and SEQ ID NO:189 has at least 99% identity to the heavy and light chains. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:188 and SEQ ID NO:189 has at least 98% identity to the heavy and light chains. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:188 and SEQ ID NO:189 has at least 97% identity to the heavy and light chains. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:188 and SEQ ID NO:189 has at least 96% identity between the heavy and light chains. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:188 and SEQ ID NO:189 has at least 95% identity to the heavy and light chains.

In one embodiment, the PD-L1 inhibitor comprises the heavy and light chain CDRs or Variable Regions (VR) of ivermectin. In one embodiment, the PD-L1 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:190, and a PD-L1 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:191 or conservative amino acid substitutions thereof. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:190 and SEQ ID NO:191 has at least 99% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:190 and SEQ ID NO:191 has at least 98% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:190 and SEQ ID NO:191 has at least 97% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:190 and SEQ ID NO:191 has at least 96% identity V _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:190 and SEQ ID NO:191 has at least 95% identity of V _H And V _L A zone.

In one embodiment, the PD-L1 inhibitor comprises a polypeptide having the sequence set forth in SEQ ID NO: 192. SEQ ID NO:193 and SEQ ID NO:194 or conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 195. SEQ ID NO:196 and SEQ ID NO:197 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on PD-L1 and/or bind to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 biological analog monoclonal antibody approved by the drug administration with reference to esvellumab. In one embodiment, the 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 the 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, which is esvellumab. 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, and the anti-PD-L1 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is esvaluzumab. anti-PD-L1 antibodies may be licensed by drug administration such as the us FDA and/or EMA of the european union. In some embodiments, the biological analog is provided as a composition further comprising one or more excipients, the one or more excipients being the same as or different from the excipients included in the reference drug or reference biological product, which is esvellumab. In some embodiments, the biological analog is provided as a composition further comprising one or more excipients, the one or more excipients being the same as or different from the excipients included in the reference drug or reference biological product, which is esvellumab.

Table 21: amino acid sequence of PD-L1 inhibitor related to Evellumab

In one embodiment, the PD-L1 inhibitor is alemtuzumab, also known as MPDL3280A or RG7446 (Roche Holding AG, basel, the subsidiary company of Switzerland Genentech, inc. Commercially available as TECENTRIQ) or an antigen-binding fragment, conjugate or variant thereof. In one embodiment, 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 its entirety). In one embodiment, the PD-L1 inhibitor is an antibody disclosed in U.S. patent application publication nos. 2010/0203056 A1, 2013/0045200 A1, 2013/0045201 A1, 2013/0045202 A1, or 2014/0065135 A1 (the disclosures of which are incorporated herein by reference in their entirety). The preparation and properties of alemtuzumab are described in U.S. patent No. 8,217,149, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequence of alemtuzumab is shown in table 22. Alemtuzumab has intra-heavy chain disulfide bonds (C23-C104) located at 22-96, 145-201, 262-322, 368-426, 22"-96", 145"-201", 262"-322" and 368 "-426"; at 23'-88', 134 '-194': 23 '"-88'" and 134 '"-194'" of a light chain disulfide bond (C23-C104); disulfide bonds within the heavy-light chains located at 221-214 'and 221 "-214'" (h 5-CL 126); heavy chain-heavy intra-chain disulfide bonds (h 11, h 14) located at 227-227 "and 230-230"; and N-glycosylation sites at 298 and 298' (HCH 2N 84.4> A).

In one embodiment, the PD-L1 inhibitor comprises SEQ ID NO:198 and SEQ ID NO:199, a light chain. In one embodiment, 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 one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:198 and SEQ ID NO:199 have heavy and light chains with at least 99% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:198 and SEQ ID NO:199 have heavy and light chains with at least 98% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:198 and SEQ ID NO:199 have heavy and light chains with at least 97% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:198 and SEQ ID NO:199 have heavy and light chains with at least 96% identity. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:198 and SEQ ID NO:199 have heavy and light chains with at least 95% identity.

In one embodiment, the PD-L1 inhibitor comprises the heavy and light chain CDRs or Variable Regions (VRs) of alemtuzumab. In one embodiment, the PD-L1 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:200, and a PD-L1 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:201 or a conservative amino acid substitution thereof. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 99% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 98% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 97% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 96% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the PD-L1 inhibitor comprises a polypeptide that is each isolated from SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 95% identity to the sequence shown in 201 _H And V _L A zone.

In one embodiment, the PD-L1 inhibitor comprises a polypeptide having the sequence set forth in SEQ ID NO: 202. SEQ ID NO:203 and SEQ ID NO:204 or conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 205. SEQ ID NO:206 and SEQ ID NO:207 or a conservative amino acid substitution of the light chain CDR1, CDR2 and CDR3 domains of the sequence depicted. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on PD-L1 and/or bind to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the anti-PD-L1 antibody is an anti-PD-L1 biological analog monoclonal antibody approved by the regulatory agency of the pharmaceutical authorities with reference to atrazumab. In one embodiment, the biological analog comprises an anti-PD-L1 antibody, the 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 the amino acid sequence of a reference drug or a reference biological product, which comprises one or more post-translational modifications as compared to the reference drug or reference biological product, the reference drug or reference biological product being alemtuzumab. 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, and the anti-PD-L1 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is alemtuzumab. anti-PD-L1 antibodies may be licensed by drug administration such as the us FDA and/or EMA of the european union. In some embodiments, the biological analog is a composition provided further comprising one or more excipients, the one or more excipients being the same as or different from the excipients included in the reference drug or reference biological product, which is alemtuzumab. In some embodiments, the biological analog is a composition provided further comprising one or more excipients, the one or more excipients being the same as or different from the excipients included in the reference drug or reference biological product, which is alemtuzumab.

Table 22: amino acid sequence of PD-L1 inhibitor related to alemtuzumab

In one embodiment, the PD-L1 inhibitor or anti-PD-L1 antibody is a remiaver Li Shan antibody or a fragment, variant, conjugate, or biological analog thereof, available from Incyte, inc.

In one embodiment, the PD-L1 inhibitor comprises some antibodies described in U.S. patent application publication No. US 2014/0341917 A1, the disclosure of which is incorporated herein by reference in its entirety. In another embodiment, antibodies that compete with any of these antibodies for binding to PD-L1 are also included. In one embodiment, the anti-PD-L1 antibody is MDX-1105 (also referred to as BMS-935559), which is disclosed in U.S. patent No. 7,943,743, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the anti-PD-L1 antibody is selected from the group of anti-PD-L1 antibodies disclosed in U.S. patent No. 7,943,743, which is incorporated herein by reference in its entirety.

In one embodiment, the PD-L1 inhibitor is a commercially available monoclonal antibody, e.g., INVITOMAB anti-m-PD-L1 clone 10F.9G2 (catalog #BE0101, bio X Cell, inc., new Hampshish, U.S.A.). In one embodiment, the anti-PD-L1 antibody is a commercially available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH 1). Some commercially available anti-PD-L1 antibodies are known to those skilled in the art.

In one embodiment, the PD-L2 inhibitor is a commercially available monoclonal antibody, such as, for example, a BIOLEGEND 24f.10c12 mouse IgG2aκ isotype (catalog #329602BIOLEGEND, inc., san diego, california), a sigma anti-PD-L2 antibody (catalog # SAB3500395, sigma-Aldrich co., san lewisi, misia) or other commercially available anti-PD-L2 antibody known to one of skill in the art.

In some embodiments, the invention includes a method of treating a cancer patient, the method comprising the step of administering a TIL regimen, wherein the TIL regimen comprises a TIL product genetically modified to express CCR, the method further comprising the step of administering a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the invention includes a composition comprising (i) a TIL product genetically modified to express CCR and (ii) a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the invention includes a kit comprising (i) a TIL product genetically modified to express CCR and (ii) a PD-1 inhibitor or a PD-L1 inhibitor.

3. Combination with CTLA-4 inhibitors

In some embodiments, the TIL therapy provided to the cancer patient may include a monotherapy TIL population therapy or may include a combination therapy including TIL and one or more CTLA-4 inhibitors.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily 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 the adaptive immune response. Similar to the T cell costimulatory protein CD28, CTLA-4 binding antigen presents CD80 and CD86 on the cell. CTLA-4 delivers an inhibitor signal to T cells, while CD28 delivers a stimulation signal. Human antibodies against human CTLA-4 have been described as immunostimulating modulators in a number of disease states, for example for the treatment or prophylaxis of viral and bacterial infections and for the treatment of cancer (WO 01/14424 and WO 00/37504). Some 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 detailed in the following paragraphs. The terms "inhibitor," "antagonist," and "blocker" are used interchangeably herein with reference to CTLA-4 inhibitors. For the avoidance of doubt, CTLA-4 inhibitors referred to herein 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.

Suitable CTLA-4 inhibitors 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), tremelimumab, anti-CD 28 antibodies, anti-CTLA-4 mucin, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the costimulatory pathway, antibodies disclosed in PCT publication WO 2001/014424, antibodies disclosed in PCT publication WO 2004/035, antibodies disclosed in US publication 2005/0201994, and antibodies disclosed in quasi-European patent publication EP 1212422 B1, the respective disclosures of which are incorporated herein by reference in their entirety. Additional CTLA-4 antibodies are described in 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; and U.S. publication nos. 2002/0039581 and 2002/086014, the disclosures of each of which are incorporated herein by reference in their entirety. Other anti-CTLA-4 antibodies useful in the methods of the invention include, for example, those disclosed in: WO 98/42752; U.S. Pat. nos. 6,682,736 and 6,207,156; hurwitz et al, proc.Natl.Acad.Sci.USA,95 (17): 10067-10071 (1998); camacho et al, j.clin.oncology,22 (145): abstract No.2505 (2004) (antibody CP-675206); mokyr et al, cancer res.,58:5301-5304 (1998) and U.S. Pat. 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 in their entirety.

Additional CTLA-4 inhibitors include, but are not limited to, the following: any inhibitor capable of disrupting the ability of CD28 antigen to bind to its cognate ligand, inhibiting CTLA-4 to bind to its cognate ligand, amplifying T cell responses by a costimulatory pathway, disrupting the ability of B7 to bind to CD28 and/or CTLA-4, disrupting the ability of B7 to activate a costimulatory pathway, disrupting the ability of CD80 to bind to CD28 and/or CTLA-4, disrupting the ability of CD80 to activate a costimulatory pathway, disrupting the ability of CD86 to bind to CD28 and/or CTLA-4, disrupting the ability of CD86 to activate a costimulatory pathway, and disrupting general costimulatory pathway activation. This necessarily includes small molecule inhibitors of other members of the costimulatory pathway of CD28, CD80, CD86, CTLA-4, and the like; antibodies to other members of the costimulatory pathway such as CD28, CD80, CD86, CTLA-4; antisense molecules directed against other members of the costimulatory pathway of CD28, CD80, CD86, CTLA-4, and the like; mucins directed against other members of the costimulatory pathway of CD28, CD80, CD86, CTLA-4, and the like; other CTLA-4 inhibitors such as RNAi inhibitors (single-and double-stranded) of other members of the costimulatory pathway such as CD28, CD80, CD86, CTLA-4.

In some embodiments, the CTLA-4 inhibitor is at 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 ^-1 M or less, 10 ^-12 M or less (e.g. between 10 ^-13 M and 10 ^-16 M) or K in any range having any of the foregoing values as endpoints _d Binding to CTLA-4. In some embodiments, the CTLA-4 inhibitor is not more than 10-fold K of ipilimumab when compared using the same assay _d Binding to CTLA-4. In some embodiments, the CTLA-4 inhibitor is about the same or less than K of ipilimumab (e.g., up to 10-fold lower or up to 100-fold lower) when compared using the same assay _d Binding to CTLA-4. In some embodiments, when compared using the same assay, the binding of CTLA-4 to CD80 or CD86 inhibited by the CTLA-4 inhibitor is integrated into the IC ₅₀ Values were higher than that mediated by ipilimumab to inhibit binding of CTLA-4 to CD80 or CD86, respectivelyMore than 10 times. In some embodiments, when compared using the same assay, the binding of CTLA-4 to CD80 or CD86 inhibited by the CTLA-4 inhibitor is integrated into the IC ₅₀ Values are about the same or less (e.g., up to 10-fold lower or up to 100-fold lower) that binding of CTLA-4 to CD80 or CD86, respectively, is inhibited by ipilimumab.

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%, 75% and 90% or 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 at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a suitable control, e.g., between 50% and 75%, 75% and 90% or 90% and 100% relative to a suitable control, by reducing binding of CTLA-4 to CD80, CD86, or both. Suitable controls in the context of assessing or quantifying the effect of an agent of interest are generally comparable biological systems (e.g., cells or subjects) that have not been exposed to (or have been exposed to a negligible amount of) an agent of interest (e.g., CTLA-4 pathway inhibitor). In some embodiments, the biological system may serve as a control for itself (e.g., the biological system may be evaluated prior to exposure or treatment with the agent and compared to a state after exposure or treatment has begun or ended). In some embodiments, a historical control may be used.

In one embodiment, the CTLA-4 inhibitor is ipilimumab (Bristol-Myers Squibb co. Commercially available 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 derived from a fully human IgG 1 kappa antibody of a transgenic mouse having human genes encoding heavy and light chains to produce a functional human reservoir. Ipilimumab may also be referred to by its CAS registry number 477202-00-9 and with reference to PCT publication WO 01/14424, which is incorporated herein by reference in its entirety for all purposes. Which is disclosed as antibody 10DI. Specifically, 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 one embodiment, the CTLA-4 inhibitor comprises an amino acid sequence consisting of SEQ ID NO:208 and the heavy chain set forth by SEQ ID NO: 209. In one embodiment, 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 one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:208 and SEQ ID NO:209 has at least 99% identity to the heavy and light chains. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:208 and SEQ ID NO:209 has at least 98% identity to the heavy and light chains. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:208 and SEQ ID NO:209 has at least 97% identity to the heavy and light chains. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:208 and SEQ ID NO:209 has at least 96% identity to the heavy and light chains. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:208 and SEQ ID NO:209 has at least 95% identity to the heavy and light chains.

In one embodiment, the CTLA-4 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of ipilimumab. In one embodiment, the CTLA-4 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:210, a CTLA-4 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:211 or conservative amino acid substitutions thereof. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:210 and SEQ ID NO:211 has at least 99% of the sequence shown in SEQ ID NO. 211V of identity _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:210 and SEQ ID NO:211 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:210 and SEQ ID NO:211 has at least 97% identity to V _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:210 and SEQ ID NO:211 has a V with at least 96% identity to the sequence shown in 211 _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:210 and SEQ ID NO:211 has at least 95% identity to V _H And V _L A zone.

In one embodiment, the CTLA-4 inhibitor comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 212. SEQ ID NO:213 and SEQ ID NO:214 or conservative amino acid substitutions thereof, and having heavy chain CDR1, CDR2 and CDR3 domains of the sequences set forth in SEQ ID NOs: 215. SEQ ID NO:216 and SEQ ID NO:217 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on CTLA-4 and/or to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the CTLA-4 inhibitor is a monoclonal antibody to a biological analog of CTLA-4 approved by the regulatory agency of the pharmaceutical industry with reference to ipilimumab. In one embodiment, the 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 the 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, which 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 shown in table 23. In some embodiments, the biological analog is an anti-CTLA-4 antibody that is authorized or filed authorized, and the anti-CTLA-4 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is ipilimumab. anti-CTLA-4 antibodies can be licensed by drug authorities such as the FDA in the united states and/or EMA in the european union. In some embodiments, the biosimilar is a composition provided as 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, which is ipilimumab. In some embodiments, the biosimilar is a composition provided as 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, which is ipilimumab.

Table 23: amino acid sequence of ipilimumab

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 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, which is administered at a dose of 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 10 mg/kg. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, and the ipilimumab is administered at a dose of about 200mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, which is administered at a dose of 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 500 mg. In some embodiments, the administration of ipilimumab 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biological analog thereof, and the ipilimumab is administered once every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the 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 up to 4 doses per 3 weeks to treat unresectable or metastatic melanoma. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered as an adjuvant treatment for melanoma. In some embodiments, ipilimumab is administered at about 10mg/kg per 3 weeks for a total of 4 doses followed by 10mg/kg per 12 weeks for up to 3 years of adjuvant treatment for melanoma. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat advanced renal cell carcinoma. In some embodiments, irinotecan is administered at about 1mg/kg every 3 weeks and a total of 4 doses of nivolumab immediately following 3mg/kg on the same day to treat advanced renal cell carcinoma. In some embodiments, after completion of the 4 doses of combination, nivolumab may be administered as a single dose according to standard dosing regimens for advanced renal cell carcinoma and/or renal cell carcinoma. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer. In some embodiments, irinotecan is administered at about 1mg/kg intravenously for 30 minutes every 3 weeks and 3mg/kg of nivolumab immediately following the same day intravenously for 30 minutes as a total of 4 doses to treat microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer. In some embodiments, nivolumab is administered as a single agent following completion of the 4-dose combination as suggested by standard dosing regimens for microsatellite high instability (MSI-H) or mismatch repair deficiency (dMMR) metastatic colorectal cancer. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat hepatocellular carcinoma. In some embodiments, irinotecan is administered as a total of 4 doses of approximately 3mg/kg intravenously every 3 weeks and 30 minutes intravenously immediately after 1mg/kg of nivolumab on the same day to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered as a single agent following standard dosing regimens for hepatocellular carcinoma after completion of the 4 dose combination. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the 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, along with 3mg/kg of nivolumab every 2 weeks, to treat metastatic non-small cell lung cancer. In some embodiments, ipilimumab is administered at about 1mg/kg every 6 weeks in combination with 360mg of nivolumab every 3 weeks and 2 cycles of platinum dual chemotherapy to treat metastatic non-small cell lung cancer. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the 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, along with 360mg of nivolumab every 3 weeks to treat malignant pleural mesothelioma. In some embodiments, the ipilimumab administration may also begin 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, the ipilimumab administration may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from the subject or patient).

Tremelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has CAS number 745013-59-6. U.S. Pat. No. 6,682,736 (incorporated herein by reference in its entirety) discloses that tramadol is antibody 11.2.1. The amino acid sequences of the heavy and light chains of tremelimumab are shown in SEQ ID NOs: 218 and SEQ ID NO:219, given in the text. Tremelimumab has been discussed in clinical trials for the treatment of various tumors including melanoma and breast cancer; wherein tremelimumab is administered intravenously as a single dose or as multiple doses in a dose range of 0.01 and 15mg/kg every 4 or 12 weeks. In the regimen provided by the invention, tremelimumab is topically and specifically administered intradermally or subcutaneously. The effective amount of tremelimumab administered intradermally or subcutaneously is typically in the range of 5 to 200 mg/dose per person. In some embodiments, the effective amount of tremelimumab is in the range of 10 to 150 mg/dose per person per dose. In some embodiments, the effective amount of tremelimumab is about 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175, or 200 mg/dose per person.

In one embodiment, the CTLA-4 inhibitor comprises an amino acid sequence consisting of SEQ ID NO:218 and the heavy chain set forth by SEQ ID NO: 219. In one embodiment, 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 one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:218 and SEQ ID NO:219, a heavy chain and a light chain having at least 99% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:218 and SEQ ID NO:219, a heavy chain and a light chain having at least 98% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:218 and SEQ ID NO:219, a heavy chain and a light chain having at least 97% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:218 and SEQ ID NO:219, a heavy chain and a light chain having at least 96% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:218 and SEQ ID NO:219, a heavy chain and a light chain having at least 95% identity.

In one embodiment, the CTLA-4 inhibitor comprises heavy and light chain CDRs or Variable Regions (VRs) of tremelimumab. In one embodiment, the CTLA-4 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:220, a CTLA-4 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:221 or conservative amino acid substitutions thereof. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:220 and SEQ ID NO:221 has at least 99% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:220 and SEQ ID NO:221 has a V with at least 98% identity to the sequence shown in 221 _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:220 and SEQ ID NO:221 has at least 97% identity to V _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:220 and SEQ ID NO:221 has a V with at least 96% identity to the sequence shown in 221 _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:220 and SEQ ID NO:221 has at least 95% identity of V _H And V _L A zone.

In one embodiment, the CTLA-4 inhibitor comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 222. SEQ ID NO:223 and SEQ ID NO:224 or conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 225. SEQ ID NO:226 and SEQ ID NO:227 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on CTLA-4 and/or to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 biological analog monoclonal antibody approved by the drug administration with reference to tremelimumab. In one embodiment, the 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 the 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, which is tremelimumab. 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 tremelimumab is shown in table 24. In some embodiments, the biological analog is an anti-CTLA-4 antibody that is authorized or filed authorized, and the anti-CTLA-4 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is tremelimumab. anti-CTLA-4 antibodies can be licensed by drug authorities such as the FDA in the united states and/or EMA in the european union. In some embodiments, the biosimilar is a composition provided as 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, which is tremelimumab. In some embodiments, the biosimilar is a composition provided as 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, which is tremelimumab.

Table 24: amino acid sequence of tremelimumab

In some embodiments, the CTLA-4 inhibitor is tremelimumab or a biological analog thereof, and tremelimumab is administered at a dose of about 0.5mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is tremelimumab or a biological analog thereof, which is administered at a dose of 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 10 mg/kg. In some embodiments, administration of tremelimumab may also begin 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 tremelimumab may also begin 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 CTLA-4 inhibitor is tremelimumab or a biological analog thereof, and tremelimumab is administered at a dose of about 200mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is tremelimumab or a biological analog thereof, which is administered at a dose of 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 500 mg. In some embodiments, administration of tremelimumab 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 tremelimumab may also begin 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 CTLA-4 inhibitor is tremelimumab or a biological analog thereof, and tremelimumab is administered once every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, administration of tremelimumab may also begin 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 tremelimumab may also begin 1, 2, or 3 weeks prior to excision (i.e., prior to obtaining a tumor sample from a subject or patient).

In one embodiment, the CTLA-4 inhibitor is za Li Fu limab from agalus or a biological analog, antigen-binding fragment, conjugate, or variant thereof. Za Li Fu monoclonal antibody is a fully human monoclonal antibody. Za Li Fu is assigned chemical abstract Company (CAS) accession number 2148321-69-9, also known as AGEN1884. The preparation and properties of za Li Fu mab are described in U.S. Pat. No. 10,144,779 and U.S. patent application publication No. US2020/0024350 A1, the disclosures of which are incorporated herein by reference in their entirety.

In one embodiment, the CTLA-4 inhibitor comprises an amino acid sequence consisting of SEQ ID NO:228 and the heavy chain given by SEQ ID NO: 229. In one embodiment, 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 one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 99% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 98% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 97% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 96% identity. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:228 and SEQ ID NO:229 has a heavy chain and a light chain with at least 95% identity.

In one embodiment, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or Variable Regions (VRs) of za Li Fu mab. In one embodiment, the CTLA-4 inhibitor heavy chain variable region (V _H ) Comprising SEQ ID NO:230, a CTLA-4 inhibitor light chain variable region (V _L ) Comprising SEQ ID NO:231 or conservative amino acid substitutions thereof. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:230 and SEQ ID NO:231 has at least 99% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:230 and SEQ ID NO:231 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:230 and SEQ ID NO:231 has at least 97% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:230 and SEQ ID NO:231 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the CTLA-4 inhibitor comprises a peptide sequence that is each of SEQ ID NO:230 and SEQ ID NO:231 sequence ofColumns have at least 95% identity V _H And V _L A zone.

In one embodiment, the CTLA-4 inhibitor comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 231. SEQ ID NO:233 and SEQ ID NO:234 or conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NOs: 235. SEQ ID NO:236 and SEQ ID NO:237 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown in fig. 237. In one embodiment, the antibody and any of the foregoing antibodies compete for binding to the same epitope on CTLA-4 and/or to the same epitope on PD-1 to which any of the foregoing antibodies binds.

In one embodiment, the CTLA-4 inhibitor is a monoclonal antibody to a biological analog of CTLA-4 approved by the drug administration reference ZA Li Fu monoclonal antibody. In one embodiment, the 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 the 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, which is za Li Fu limab. 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 za Li Fu mab is shown in table 25. In some embodiments, the biological analog is an anti-CTLA-4 antibody that is licensed or otherwise applied for authorization, and the anti-CTLA-4 antibody is provided in a different formulation than the formulation of the reference drug or reference biologic, which is za Li Fu mab. anti-CTLA-4 antibodies can be licensed by drug authorities such as the FDA in the united states and/or EMA in the european union. In some embodiments, a biological analog 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 biological product, which is za Li Fu mab. In some embodiments, a biological analog 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 biological product, which is za Li Fu mab.

Table 25: amino acid sequence of Za Li Fu monoclonal antibody

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 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent publications: US 2019/0048096 A1; US 2020/0223907; US 2019/0201334; US 2019/0201334; 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, each of which is incorporated herein by reference in its entirety. Additional CTLA-4 antibodies are described in 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 is incorporated by reference herein in its entirety). 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); camahho et al, J.Clin.Oncol.,2004,22,145 (Abstract No.2505 (2004) (antibody CP-675206), or Mokyr et al, cancer Res.,1998,58,5301-5304 (1998), each of which is incorporated herein by reference in its entirety.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand as disclosed in WO 1996/040915 (incorporated herein by reference in its entirety).

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, anti-CTLA-4 RNAi molecules can be found in PCT publications WO 1999/032619 and WO 2001/029058; U.S. publications 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, 2005/0100913, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443; and/or forms of the molecules described in U.S. Pat. nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference in their entirety). In certain instances, 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 its entirety). In certain instances, the anti-CTLA-4 RNAi molecules are in the form of double stranded RNAi molecules described in U.S. patent nos. 7,056,704 and 7,078,196 (incorporated herein by reference in their entirety). In some embodiments, the CTLA-4 inhibitor is an aptamer described in international patent application publication WO 2004/081021 (incorporated herein by reference in its entirety).

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 EP 0928290 (incorporated herein by reference in its entirety).

In some embodiments, the invention includes a method of treating a cancer patient, the method comprising the step of administering a TIL regimen, wherein the TIL regimen comprises a TIL product genetically modified to express CCR, the method further comprising the step of administering a CTLA-4 inhibitor. In some embodiments, the invention includes a composition comprising (i) a TIL product genetically modified to express CCR and (ii) a CTLA-4 inhibitor. In some embodiments, the invention includes a kit comprising (i) a TIL product genetically modified to express CCR and (ii) a CTLA-4 inhibitor.

In some embodiments, the invention includes a method of treating a cancer patient, the method comprising the step of administering a TIL regimen comprising a TIL product genetically modified to express CCR, the method further comprising the step of administering a CTLA-4 inhibitor and a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the invention includes a composition comprising (i) a TIL product genetically modified to express CCR, (ii) a CTLA-4 inhibitor, and (iii) a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the invention includes a kit comprising (i) a TIL product genetically modified to express CCR, (ii) a CTLA-4 inhibitor, and (iii) a PD-1 inhibitor or a PD-L1 inhibitor.

4. Pretreatment for lymphocyte depletion in patient

In one embodiment, the invention includes a method of treating cancer with a population of TILs, the patient being pre-treated with non-myeloablative chemotherapy prior to infusion of a TIL according to the present disclosure. In one embodiment, the invention includes a TIL population for treating cancer in a patient that has been pretreated with non-myeloablative chemotherapy. In one embodiment, the TIL population is for infusion administration. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (27 and 26 days prior to TIL infusion) and fludarabine 25mg/m ² Day 5 (days 27 to 23 before TIL infusion). In one embodiment, following non-myeloablative chemotherapy and TIL infusion according to the present disclosure (day 0), the patient receives 720,000IU/kg intravenous IL-2 (aldinterleukin, commercially available as PROLEUKIN) every 8 hours for intravenous infusion to physiological tolerance. In certain embodiments, the TIL population is used in combination with IL-2 to treat cancer, and IL-2 is administered after the TIL population.

Experiments have found that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by clearing regulatory T cells and competing elements of the immune system ("cytokine repertoire"). Thus, some embodiments of the invention subject the patient to a lymphocyte depletion step (sometimes also referred to as "immunosuppressive conditioning") prior to introducing the TIL of the invention.

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 immunol. Immunther. 2011,60,75-85, muranski et al, nat. Clin. Practice. Oncol.,2006,3,668-681, dudley et al, J. Clin. Oncol.2008,26,5233-5239, and Dudley et al, J. Clin. Oncol.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 treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more than 7 days. 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, a concentration of 0.5 μg/mL to 10 μg/mL of maphosphamide (the active form of cyclophosphamide) is obtained by administering cyclophosphamide. In some embodiments, a concentration of 1 μg/mL of maphos-mide (the active form of cyclophosphamide) is obtained by administering cyclophosphamide. 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 than 7 days. In some embodiments, cyclophosphamide is present at 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 Dosage per 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 at 250mg/m ² Administration is carried out for 4 to 5 days. In some embodiments, cyclophosphamide treatment is at 250mg/m ² Administration was performed i.v. for 4 days.

In some embodiments, lymphocyte depletion is performed by administering fludarabine and cyclophosphamide together to the patient. In some embodiments, fludarabine is present at 25mg/m ² Applied/day i.v. and cyclophosphamide at 2 50mg/m ² Administration was performed i.v. for 4 days.

In one embodiment, lymphocyte depletion is achieved by administration of cyclophosphamide at a dose of 60mg/m ² Two days per day followed by administration of fludarabine at a dose of 25mg/m ² Five days per day.

In one embodiment, lymphocyte depletion is achieved by administering a dose of 60mg/m ² Cyclophosphamide per day for two days and at an administration dose of 25mg/m ² The day's fludarabine is performed for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days and lymphocyte depletion is performed for five days in total.

In one embodiment, lymphocyte depletion is achieved by administering a dose of about 50mg/m ² Cyclophosphamide per day for two days and at an administration dose of about 25mg/m ² The day's fludarabine is performed for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days and lymphocyte depletion is performed for five days in total.

In one embodiment, lymphocyte depletion is achieved by administering a dose of about 50mg/m ² Cyclophosphamide per day for two days and at a dosage of about 20mg/m ² The day's fludarabine is performed for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days and lymphocyte depletion is performed for five days in total.

In one embodiment, lymphocyte depletion is achieved by administering a dose of about 40mg/m ² Cyclophosphamide per day for two days and at a dosage of about 20mg/m ² The day's fludarabine is performed for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days and lymphocyte depletion is performed for five days in total.

In one embodiment, lymphocyte depletion is achieved by administering a dose of about 40mg/m ² Cyclophosphamide per day for two days and at a dose of about 15mg/m ² The day's fludarabine is performed for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days and lymphocyte depletion is performed for five days in total.

In one embodiment, lymphocyte depletion is achieved by administering a dose of 60mg/m ² Ring for dayPhosphoramides and dose of 25mg/m ² Fludarabine per day for two days and then administered at a dose of 25mg/m ² Fludarabine/day for three days.

In one embodiment, cyclophosphamide is administered with mesna. In one embodiment, mesna is administered at 15 mg/kg. In one embodiment, when mesna is infused and if infused continuously, mesna can be infused with cyclophosphamide within about 2 hours (on day-5 and/or-4), followed by infusion at a rate of 3 mg/kg/hour for the remaining 22 hours within 24 hours beginning simultaneously with each cyclophosphamide dose.

In one embodiment, lymphocyte depletion further comprises the step of beginning treatment of the patient with the IL-2 regimen the next day after administration of the third TIL population to the patient.

In one embodiment, lymphocyte depletion further comprises the step of beginning treatment of the patient with the IL-2 regimen on the same day as the third TIL population is administered to the patient.

In some embodiments, lymphocyte depletion comprises 5 days of pretreatment therapy. In some embodiments, the number of days is indicated as-5 to-1 or 0 to 4. In some embodiments, the regimen comprises cyclophosphamide at day-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 Intravenous fludarabine. In some embodiments, the regimen further comprises 25mg/m on days-5 and-1 (i.e., days 0 to 4) ² Intravenous fludarabine. In some embodiments, the regimen further comprises 25mg/m on days-5 and-1 (i.e., days 0 to 4) ² Intravenous fludarabine.

In some embodiments, the non-myeloablative lymphocyteThe depletion regimen included administration of a dose of 60mg/m ² Cyclophosphamide per day and dose 25mg/m ² Fludarabine per day for two days and then administered at a dose of 25mg/m ² Five days total of steps per day of fludarabine.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering cyclophosphamide at a dose of 60mg/m 2/day and cyclophosphamide at a dose of 25mg/m ² Fludarabine per day for two days and then administered at a dose of 25mg/m ² The method comprises the step of carrying out three days of fludarabine per day.

In some embodiments, the non-myeloablative lymphocyte depletion regimen is administered according to table 26.

Table 26: 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 (if needed)	X	X
											Fludarabine 25mg/m ² Day/day	X	X	X	X	X
TIL infusion						X

In some embodiments, the non-myeloablative lymphocyte depletion therapy is administered according to table 27.

Table 27: 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 (if needed)

X

Fludarabine 25mg/m ² Day/day

X

TIL infusion

X

In some embodiments, the non-myeloablative lymphocyte depletion therapy is administered according to table 28.

Table 28: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 60mg/kg

X

Mesna (if needed)

X

Fludarabine 25mg/m ² Day/day

X

TIL infusion

X

In some embodiments, the non-myeloablative lymphocyte depletion therapy is administered according to table 29.

Table 29: 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 (if needed)	X	X
											Fludarabine 25mg/m ² Day/day			X	X	X
TIL infusion						X

In some embodiments, the non-myeloablative lymphocyte depletion therapy is administered according to table 30.

Table 30: 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 (if needed)	X	X
											Fludarabine 30mg/m ² Day/day	X	X	X	X	X
TIL infusion						X

In some embodiments, the non-myeloablative lymphocyte depletion therapy is administered according to table 31.

Table 31: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')

-4

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300mg/kg

X

Mesna (if needed)

X

Fludarabine 30mg/m ² Day/day

X

TIL infusion

X

In some embodiments, the non-myeloablative lymphocyte depletion therapy is administered according to table 32.

Table 32: exemplary lymphocyte depletion and treatment regimens

Tiantian (Chinese character of 'Tian')

-3

-2

-1

0

1

2

3

4

Cyclophosphamide 300mg/kg

X

Mesna (if needed)

X

Fludarabine 30mg/m ² Day/day

X

TIL infusion

X

In some embodiments, the non-myeloablative lymphocyte depletion therapy is administered according to table 33.

Table 33: 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 (if needed)	X	X
											Fludarabine 30mg/m ² Day/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 TIL composition described herein, including TIL products genetically modified to express CCR as described herein, and can also include infusion MILs and PBLs in place of TIL infusion, as well as addition of IL-2 regimens and administration of co-therapies (e.g., PD-1 and PD-L1 inhibitors) as described herein.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering melphalan (melphalan) to a total dose of 100mg/m over a 1, 2, or 3 day period prior to the day of TIL infusion ² . In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering melphalan to a total dose of 200mg/m over a 1, 2, or 3 day period prior to the day of TIL infusion ² . In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering melphalan to a total dose of 100mg/m over a 1, 2, or 3 day period prior to the day of TIL infusion ² And fludarabine at 30mg/m ² Dosage per day. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering melphalan to a total dose of 200mg/m over a 1, 2, or 3 day period prior to the day of TIL infusion ² And fludarabine at 30mg/m ² Dosage per day.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering an anti-CD 45 antibody. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering an anti-CD 45 antibody-drug conjugate. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering an anti-CD 45 antibody-radioisotope conjugate. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering apamizumab- ¹³¹ I. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering apamizumab at a dose of 25mCi, 50mCi, 75mCi, 100mCi, 150mCi, or 200mCi between 2 and 9 days prior to TIL infusion ¹³¹ I. In some embodiments, notThe myeloablative lymphocyte depletion regimen involves the administration of apamizumab- ¹³¹ I. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering apamizumab- ¹³¹ I. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering apamizumab at a dose of about 75mCi about 6 days prior to TIL infusion ¹³¹ I. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering apamizumab at a dose of about 100mCi about 7 days prior to TIL infusion ¹³¹ I。

In some embodiments, the TIL infusion used with the foregoing embodiments of the myeloablative lymphocyte depletion regimen can be any TIL composition described herein, including TIL products genetically modified to express CCR as described herein, can also include infusion MILs and PBLs in place of TIL infusion, as well as adding alternative lymphocyte depletion regimens, including anti-CD 52 antibody alemtuzumab or variants, fragments, antibody-drug conjugates, or biological analogs thereof.

IL-2 protocol

In one embodiment, the IL-2 regimen comprises a high dose IL-2 regimen comprising intravenous administration of the aldesleukin or a biological analog or variant thereof beginning the next day after administration of the therapeutically effective portion of the therapeutic TIL population, the aldesleukin or a biological analog or variant thereof being administered at a dose of 0.037mg/kg or 0.044mg/kg IU/kg (patient body mass) using 15 minutes bolus intravenous infusion every eight hours until tolerated, up to 14 doses. After 9 days of rest, this time course can be repeated for additional 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 one embodiment, the IL-2 regimen comprises a taper IL-2 regimen. The taper IL-2 protocol has been described in O' Day et al, J.Clin.Oncol.1999,17,2752-61 and Eton et al, cancer 2000,88,1703-9, the disclosures of which are incorporated herein by reference in their entirety. In one embodiment, the decreasing IL-2 regimen comprisesIntravenous administration 18×10 over 6 hours ⁶ IU/m ² Alterleukin or a biological analogue or variant thereof, followed by intravenous administration of 18X 10 over a period of 12 hours ⁶ IU/m ² Followed by intravenous administration of 18 x 10 over 24 hours ⁶ IU/m ² Followed by intravenous administration of 4.5X10 in 72 hours ⁶ IU/m ² . This treatment cycle may be repeated every 28 days for up to four cycles. In one embodiment, the decreasing IL-2 regimen includes day 1 18,000,000IU/m ² Day 29,000,000IU/m ² 4,500,000IU/m on days 3 and 4 ² 。

In one embodiment, 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, nat.immunology 2000,19,665-673; hartemann et al, lancet Diabetes Endocrinol.2013,1,295-305; and the low dose IL-2 regimen described in Rosenzwaig et al, ann. Rheum. Dis.2019,78,209-217, the disclosures of which are incorporated herein by reference in their entirety. In one embodiment, the low dose IL-2 regimen comprises administration every m every 24 hours ² 18×10 ⁶ Aldi interleukin of IU or a biological analogue or variant thereof as continuous infusion for 5 days, followed by 2 to 6 days without IL-2 therapy, optionally followed by an additional 5 days every 24 hours every m ² 18×10 ⁶ Intravenous aldesleukin or a biological analogue or variant thereof of IU is optionally not used for IL-2 therapy for the following 3 weeks as a continuous infusion, after which additional cycles may be administered.

In one embodiment, 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 one embodiment, the IL-2 regimen comprises administering Bei Jiade Lu Jin, 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 one embodiment, 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 one embodiment, the IL-2 regimen comprises administering endo-tile Lu Jin alpha 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 one embodiment, the IL-2 regimen comprises administering an IL-2 fragment that is implanted onto the backbone of the antibody. In one embodiment, the IL-2 regimen comprises administering an antibody cytokine implant protein that binds to a low affinity receptor for IL-2. In one embodiment, the antibody cytokine implant protein comprises a heavy chain variable region (V _H ) Light chain variable region (V) _L ) And implant into V _H Or V _L The heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, HCDR3, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, or a fragment thereof, wherein the antibody cytokine implant protein preferentially amplifies T effector cells over regulatory T cells. In one embodiment, the antibody cytokine implant protein comprises a heavy chain variable region (V _H ) Light chain variable region (V) _L ) And implant into V _H Or V _L The heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, HCDR3, and the light chain variable region comprises LCDR1, LCDR2, LCDR3, or a fragment thereof, wherein the IL-2 molecule is a mutein and the antibody cytokine implantation protein preferentially amplifies T effector cells over regulatory T cells. In one embodiment, the IL-2 regimen comprises administering an antibody or fragment, variant or biological analog thereof comprising a polypeptide 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 antibody cytokine implant proteins described herein have a higher affinity than wild-type IL-2 molecules such as, but not limited to, aldesleukin Or a longer serum half-life of the comparable molecule.

In one embodiment, the IL-2 regimen comprises administering an IL-2 fragment that is implanted onto the backbone of the antibody. In one embodiment, the IL-2 regimen comprises administering an antibody cytokine implant protein that binds to a low affinity receptor for IL-2. In one embodiment, the IL-2 regimen comprises administering an antibody cytokine implant protein that does not have an effect of binding to the IL-2Rα receptor and exhibits enhanced binding to IL-2Rβ and/or IL-2Rγ receptor compared to aldesleukin.

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, and can also include infusion of MILs and PBLs in place of TIL infusion, as well as addition of IL-2 regimens and administration of co-therapies (e.g., PD-1 and PD-L1 inhibitors) as described herein.

In some embodiments, the invention includes a method of treating a cancer patient, the method comprising the step of administering a TIL regimen, wherein the TIL regimen comprises a TIL product genetically modified to express CCR, the method further comprising the step of administering an IL-2 regimen. In some embodiments, the invention includes a composition comprising (i) a TIL product genetically modified to express CCR and (ii) an IL-2 regimen. In some embodiments, the invention includes a kit comprising (i) a TIL product genetically modified to express CCR and (ii) an IL-2 regimen.

In some embodiments, the invention includes a method of treating a cancer patient, the method comprising the step of administering a TIL regimen, wherein the TIL regimen comprises a TIL product genetically modified to express CCR, the method further comprising the step of administering an IL-2 regimen and a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the invention includes a composition comprising (i) a TIL product genetically modified to express CCR, (ii) an IL-2 regimen, and (iii) a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the invention includes a kit comprising (i) a TIL product genetically modified to express CCR, (ii) an IL-2 regimen, and (iii) a PD-1 inhibitor or a PD-L1 inhibitor.

In some embodiments, the invention includes a method of treating a cancer patient, the method comprising the step of administering a TIL regimen, wherein the TIL regimen comprises a TIL product genetically modified to express CCR, the method further comprising the step of administering a CTLA-4 inhibitor and an IL-2 regimen. In some embodiments, the invention includes a composition comprising (i) a TIL product genetically modified to express CCR, (ii) a CTLA-4 inhibitor, and (iii) an IL-2 regimen. In some embodiments, the invention includes a kit comprising (i) a TIL product genetically modified to express CCR, (ii) a CTLA-4 inhibitor, and (iii) an IL-2 regimen.

In some embodiments, the invention includes a method of treating a cancer patient, the method comprising the step of administering a TIL regimen, wherein the TIL regimen comprises a TIL product genetically modified to express CCR, the method further comprising the step of administering an IL-2 regimen, a CTLA-4 inhibitor, and a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the invention includes a composition comprising (i) a TIL product genetically modified to express CCR, (ii) an IL-2 regimen, (iii) a PD-1 inhibitor or a PD-L1 inhibitor, and (iii) a CTLA-4 inhibitor. In some embodiments, the invention includes a kit comprising (i) a TIL product genetically modified to express CCR, (ii) an IL-2 regimen, (iii) a PD-1 inhibitor or a PD-L1 inhibitor, and (iii) a CTLA-4 inhibitor.

Chimeric costimulatory receptors

In some embodiments, the foregoing manufacturing processes for manufacturing TILs, MILs, and PBLs, including generation 2 and generation 3, and other processes, can be modified to include steps comprising viral or non-viral transduction of TILs, MILs, or PBLs to express more than one CCR as described herein. In one embodiment, the CCR comprises an extracellular binding domain and an intracellular signaling domain. In one embodiment, the CCR comprises an extracellular binding domain and one or more intracellular signaling domains. In one embodiment, the CCR comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the CCR comprises an extracellular binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In one embodiment, CCR is a single polypeptide containing multiple linked domains. In some embodiments, CCR is a switch receptor. In some embodiments, the CCR comprises one or more polypeptide domains as described in U.S. patent application publication No. US 2019/0388468 A1, the disclosure of which is incorporated herein by reference in its entirety. In other embodiments, the CCR comprises one or more polypeptide domains as described in international patent application publication No. WO 2020/152451 A1, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, CCRs of the present invention used in combination with the TIL, MIL, or PBL manufacturing processes described herein have the format shown in FIG. 34. In some embodiments, the CCR of the present invention used in combination with the TIL, MIL, or PBL manufacturing processes described herein comprise the domains shown in fig. 34, operably linked to each other as shown in fig. 34.

A. Extracellular domain

In one embodiment, the CCR comprises an extracellular domain. In one embodiment, the CCR comprises an extracellular domain that binds to a tumor-associated protein. In one embodiment, the extracellular domain binds to a tumor-associated cell surface molecule. In one embodiment, the extracellular domain binds to a tumor-associated extracellular molecule. In one embodiment, the extracellular domain binds to a tumor-associated antigen. In one embodiment, the extracellular domain binds to PD-L1, also known as CD274 and is encoded by PDCD 1. In one embodiment, the extracellular domain is a PD-1 domain that binds to PD-L1 (also referred to as CD 274). In one embodiment, the extracellular domain binds to a tumor-associated antigen, which is a neoantigen. In one embodiment, the extracellular domain binds to a tumor-associated antigen, which is a peptide-major histocompatibility complex. In one embodiment, the extracellular domain binds to a tumor-associated antigen, which is a heat shock protein peptide complex. In one embodiment, the extracellular domain binds to a protein selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, FR alpha, EPCAM (CD 326), PD-1, PD-L1 (CD 274), PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, and MUC 16.

In some embodiments, extracellular binding comprises an scFv capable of binding to a tumor associated antigen. In some embodiments, the scFv comprises a polypeptide capable of binding to a polypeptide selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, and CD228. Protein-bound V of LRRC15, CEA, FR alpha, EPCAM (CD 326), PD-1, PD-L1 (CD 274), PSMA, gp100, MUC1, MCSP, EGFR, GD, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, B7-H3 and MUC16 _H And V _L A chain. In some embodiments, the invention includes modifications of the scFv amino acid sequences disclosed herein to produce functionally equivalent molecules, such as conservative amino acid substitutions. For example, V of scFv binding domain contained within CCR _H Or V _L Can be modified to retain the original V with scFv _H Or V _L The framework regions are at least about 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% identical. The invention also includes modifications of the complete CCR construct at more than one amino acid sequence of the various domains of the CCR construct to produce functionally equivalent molecules.

In one embodiment, the extracellular domain binds to a human tumor-associated antigen. In one embodiment, the extracellular domain binds to a murine tumor-associated cell surface molecule.

In some embodiments, the extracellular domain is at a K of about 100pM or less _D Bind human or murine tumor associated antigen at a K of about 90pM or less _D Bind human or murine tumor associated antigen at a K of about 80pM or less _D Bind human or murine tumor associated antigen at a K of about 70pM or less _D Bind human or murine tumor associated antigen at a K of about 60pM or less _D Bind human or murine tumor associated antigen at a K of about 50pM or less _D Bind human or murine tumor associated antigen at a K of about 40pM or less _D Bind human or murine tumor associated antigen at a K of about 30pM or less _D Bind human or murine tumor associated antigen at a K of about 20pM or less _D Bind to human or murine tumor associated antigen or at a K of about 10pM or less _D Bind to human or murine tumor-associated antigens.

In some embodiments, the cellsThe ectodomain is at about 7.5X10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine tumor associated antigen at about 7.5X10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine tumor associated antigen at about 8X 10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine tumor associated antigen at about 8.5X10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine tumor associated antigen at about 9X 10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine tumor associated antigen at about 9.5X10 ⁵ K of 1/M.s or more _{Association with} Binding to human or murine tumor associated antigen or at about 1X 10 ⁶ K of 1/M.s or more _{Association with} Binding to human or murine tumor-associated antigens.

In some embodiments, the extracellular domain is present in about 2×10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.1X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.2X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.3X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.4X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.5X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.6X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.7X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.8X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen at about 2.9X10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor associated antigen or at about 3X 10 ^-5 K of 1/s or less _{Dissociation of} Binding to human or murine tumor-associated antigens.

Adapted to be used in conjunction with V as described herein _H And V _L Domain as CCR cellThe linker sequences of the outer scFv domains are given in table 34. In one embodiment, a CCR of the invention comprises an extracellular domain comprising an scFv comprising a V linked by a linker sequence _H Binding domains and V _L Binding domain. In some embodiments, the scFv of the invention has the following format: (V) _H ) - (scFv linker) - (V) _L ) - (the remainder of the construct). In some embodiments, the scFv of the invention has the following format: (V) _L ) - (linker) - (V) _H ) - (the remainder of the construct). In some embodiments, the linker is selected from the linkers given in table 34. In some embodiments, the linker is SEQ ID NO:238 or a conservative amino acid substitution thereof or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:239 or a conservative amino acid substitution thereof or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:240 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:241 or conservative amino acid substitutions thereof or sequences having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:242 or a conservative amino acid substitution thereof or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:243 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto.

Table 34: amino acid sequence of scFv linker sequence

Adapted to be used in conjunction with V as described herein _H And V _L The domain is given in table 8 as an additional linker sequence for the extracellular scFv domain of CCR. In one placeIn some embodiments, the linker is SEQ ID NO:63 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:64 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:65 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:66 or a conservative amino acid substitution thereof or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:67 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:68 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:69 or a conservative amino acid substitution thereof or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:70 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:71 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:72 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto.

V suitable for collocating scFv described herein _H And V _L The additional linker sequences for the domains as extracellular domains of CCR are given in table 9. In some embodiments, the linker is SEQ ID NO:74 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodimentsWherein the linker is SEQ ID NO:75 or a conservative amino acid substitution thereof, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto. In some embodiments, the linker is SEQ ID NO:76 or a conservative amino acid substitution thereof or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95% homology thereto.

Alternative linker sequences for scFv domains suitable for construction of the extracellular domain of CCR are described in Bird et al, science 1988,242,423-426, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the CCR of the present invention comprises a signal peptide. Without being bound by theory, CCR comprising a signal peptide may be directed to the endoplasmic reticulum upon intracellular expression and subsequently to the cell surface where it is expressed. In one embodiment, the signal peptide may be located at the amino terminus of CCR. Any suitable signal peptide known in the art may be used with the CCR of the present invention, such as those described in U.S. patent No. 9,856,322 and U.S. patent application publication nos. US 2019/03251404 A1, US 2019/0002573 A1, US 2020/0024342 A9 and US 2020/0078399 A1, the disclosures of which are incorporated herein by reference in their entirety. Other suitable signal peptides are described elsewhere herein, including in the examples.

1. Extracellular PD-1 domains

In one embodiment, a CCR of the invention comprises an extracellular domain comprising a PD-1 domain. In one embodiment, the CCR of the present invention comprises a fusion protein comprising an extracellular domain, a transmembrane domain and an intracellular domain, the extracellular domain being at least a portion of an extracellular domain of an inhibitory polypeptide, such as PD-1, associated with a negative signal that prevents activation of an immune response or induces apoptosis of a TIL population, the intracellular domain being at least a portion of an intracellular domain of a stimulatory polypeptide associated with a positive signal, such as CD28, that activates an immune cell, and further, capable of converting a negative signal in an immune cell to the positive signal when the fusion protein is displayed on the cell to convert the negative immune response to a positive immune response.

The amino acid sequences of exemplary PD-1 domains are provided in table 35. An exemplary PD-1 CCR construct using these domains is shown in figure 35. In one embodiment, the CCR of the present invention comprises an extracellular PD-1 domain as shown in fig. 35 or fig. 36. PD-1 domains and CCR constructs using such domains (also referred to as PD-1 switch CCR constructs), including methods of making, characterizing, and using the same, are also described in U.S. patent application publication No. US 2019/0345219 A1, the disclosure of which is incorporated herein by reference in its entirety. In embodiments of the invention, the PD-1 switch CCR construct is transduced into TIL during the generation 2, generation 3 or other TIL production process, including during the period between the prep and REP phases of the generation 2 process.

In one embodiment, a CCR of the invention comprises an extracellular domain comprising the amino acid sequence of SEQ ID NO:244 or a conservative amino acid substitution thereof. In one embodiment, a CCR of the invention comprises an extracellular domain comprising the amino acid sequence of SEQ ID NO:244 or a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% homology thereto.

In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain comprising the amino acid sequence of SEQ ID NO:245 or a conservative amino acid substitution thereof. In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain comprising the amino acid sequence of SEQ ID NO:245 or a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% homology thereto.

In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain comprising the amino acid sequence of SEQ ID NO:246 or conservative amino acid substitutions thereof. In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain comprising the amino acid sequence of SEQ ID NO:246 or a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% homology thereto.

Table 35: amino acid sequence of exemplary PD-1 extracellular Domain

Nucleotide sequences encoding exemplary PD-1 domains are provided in table 36. In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain comprising a sequence consisting of SEQ ID NO:247, and a PD-1 domain encoded by the nucleotide sequence of 247. In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain consisting of a polypeptide comprising a sequence that retains SEQ ID NO:247, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical. In embodiments including the foregoing embodiments, the nucleotide sequences in table 36 are codon optimized to improve protein expression.

In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain comprising a sequence consisting of SEQ ID NO:248 to the PD-1 and CD28 domains. In one embodiment, a CCR of the invention comprises an extracellular and transmembrane domain consisting of a polypeptide comprising a sequence that retains SEQ ID NO:248, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical. In embodiments including the foregoing embodiments, the nucleotide sequences in table 36 are codon optimized to improve protein expression.

Table 36: nucleotide sequences of selected exemplary extracellular PD-1 domains

In one embodiment, the CCR of the present invention comprises a PD-1 switch construct as described in Liu et al, cancer Res.2016,76,1578-90, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the CCR of the present invention comprises a PD-1 switch construct as described in international patent application publication No. WO 2018/119298 A1, the disclosure of which is incorporated herein by reference in its entirety.

2. Extracellular PD-L1 binding domains

In one embodiment, a CCR of the invention comprises an extracellular domain comprising a PD-L1 binding scFv domain. In one embodiment, a CCR of the invention comprises an extracellular anti-PD-L1 domain comprising V _H Domain and V _L A domain. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-PD-L1V _H And V _L Domains, linkers are as described herein. In one embodiment, the anti-PD-L1 scFv domain comprises scFv antibodies 38A1 and 19H9, the nature and preparation of which (including the nucleotide sequences encoding the antibodies) are described in U.S. patent application publication No. US 2019/0298770 A1, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary anti-PD-L1 binding scFv domains are provided in table 37. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-PD-L1V _H And V _L Domain, V _H The domain is selected from SEQ ID NO: 250. SEQ ID NO:259 and conservative amino acid substitutions thereof, V _L The domain is selected from SEQ ID NO: 251.SEQ ID NO:260 and conservative amino acid substitutions thereof.

Table 37: amino acid sequence of exemplary extracellular PD-L1 binding Domain

In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:249 or conservative amino acid substitutions thereof. In one embodiment, the anti-PD-L1 scFv domain comprises scFv antibody 38A1 or a conservative amino acid substitution thereof. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 99% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 98% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 97% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 96% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 95% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 90% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 85% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:249 has an scFv domain with at least 80% identity.

In one embodiment, the anti-PD-L1 scFv domain comprises a heavy chain variable region (V _H ) Domain and light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:250 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:251 or conservative amino acid substitutions thereof. In one embodiment, anti-PD-L1The scFv domain comprises the sequence of each of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:250 and SEQ ID NO:251 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-PD-L1 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 252. SEQ ID NO:253 and/or SEQ ID NO:254, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 255. SEQ ID NO:256 and/or SEQ ID NO:257, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:258 or a conservative amino acid substitution thereof. In one embodiment, the anti-PD-L1 scFv domain comprises scFv antibody 19H9 or a conservative amino acid substitution thereof. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 99% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 98% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 97% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 96% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 95% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 90% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 85% identity. In one embodiment, the anti-PD-L1 scFv domain comprises a sequence that hybridizes to SEQ ID NO:258 has an scFv domain with at least 80% identity.

In one embodiment, the anti-PD-L1 scFv domain comprises a heavy chain variable region (V _H ) Domain and light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:259 or conservative amino acid substitution thereof, light chain variable region (V _L ) Comprising SEQ ID NO:260 or conservative amino acid substitutions thereof. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, anti-PD-L1 scFv domains comprise the respective amino acid sequences of SEQ ID NOs: 259 and SEQ ID NO:260 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:259 and SEQ ID NO:260 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-PD-L1 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 261. SEQ ID NO:262 and/or SEQ ID NO:263, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 264. SEQ ID NO:265 and/or SEQ ID NO:266 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequence depicted.

In one embodiment, the anti-PD-L1 scFv domain comprises a heavy chain variable region of dewaruzumab (V _H ) Domain and light chain variable region (V _L ) A domain. In one embodiment, the anti-PD-L1 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:180 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:181 or conservative amino acid substitutions thereof. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has a V with at least 98% identity to the sequence shown in figure 181 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has a V with at least 97% identity to the sequence shown in figure _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has a V with at least 96% identity to the sequence shown in 181 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has a V with at least 95% identity to the sequence shown in figure 181 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has a V with at least 90% identity to the sequence shown in figure 181 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has a V with at least 85% identity to the sequence shown in figure 181 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:180 and SEQ ID NO:181 has a V with at least 80% identity to the sequence shown in figure 181 _H And V _L A zone.

In one embodiment, the anti-PD-L1 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 182. SEQ ID NO:183 and/or SEQ ID NO:184, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 185. SEQ ID NO:186 and/or SEQ ID NO:187, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-PD-L1 scFv domain comprises the heavy chain variable region (V _H ) Domain and light chain variable region (V _L ) A domain. In one embodiment, the anti-PD-L1 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:190 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:191 or conservation thereof Amino acid substitutions. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 99% identity to the sequence shown in 191 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 98% identity to the sequence shown in 191 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 97% identity to the sequence shown in 191 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 96% identity to the sequence shown in 191 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 95% identity to the sequence shown in 191 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 90% identity to the sequence shown in 191 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 85% identity to the sequence shown in 191 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:190 and SEQ ID NO:191 has a V with at least 80% identity to the sequence shown in 191 _H And V _L A zone.

In one embodiment, the anti-PD-L1 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 192. SEQ ID NO:193 and/or SEQ ID NO:194, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 195. SEQ ID NO:196 and/or SEQ ID NO:197 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown.

In one embodiment, the anti-PD-L1 scFv domain comprises the heavy chain variable region (V _H ) Domain and light chain variable region (V _L ) A domain. In one embodiment, the anti-PD-L1 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:200 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:201 or a conservative amino acid substitution thereof. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 99% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 98% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 97% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 96% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 95% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 90% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 85% identity to the sequence shown in 201 _H And V _L A zone. In one embodiment, the anti-PD-L1 scFv domain comprises the amino acid sequence of SEQ ID NO:200 and SEQ ID NO:201 has a V with at least 80% identity to the sequence shown in 201 _H And V _L A zone.

In one embodiment, the anti-PD-L1 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 202. SEQ ID NO:203 and/or SEQ ID NO:204 or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 205. SEQ ID NO:206 and/or SEQ ID NO:207 or a conservative amino acid substitution of the light chain CDR1, CDR2 and CDR3 domains of the sequence depicted.

In one embodiment, the anti-PD-L1 binding domain comprises scFv, V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a conservative amino acid substitution thereof, or a nucleotide encoding such a sequence, as disclosed in U.S. patent application publication No. US 2019/0048085 A1, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-PD-L1 binding domain comprises scFv, V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a conservative amino acid substitution thereof, or a nucleotide encoding such a sequence, as disclosed in U.S. patent No. 10,604,581, the disclosure of which is incorporated herein by reference in its entirety.

3. Extracellular CEA binding domains

In one embodiment, the CCR of the present invention comprises an extracellular domain comprising a carcinoembryonic antigen (CEA) binding domain, also referred to herein as an anti-CEA domain. In one embodiment, a CCR of the invention comprises an extracellular domain comprising a CD66 binding domain. In one embodiment, a CCR of the present invention comprises an extracellular domain comprising a CD66 binding domain selected from the group consisting of a CD66a binding domain, a CD66b binding domain, a CD66c binding domain, a CD66d binding domain, a CD66e binding domain and a CD66f binding domain. In one embodiment, the CEA or CD66 binding domain is a scFv domain. In one embodiment, the CEA binding domain binds to murine CEA. In one embodiment, the CEA binding domain binds to human CEA. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-CEA V _H And V _L Domains, linkers are as described herein. In one embodiment, the CEA binding domain comprises CDRs, V, described in U.S. Pat. No. 8,470,994 _H And V _L Domain-prepared scFv antibodies, the disclosure of which is incorporated by referenceThe body is incorporated herein. The amino acid sequences of exemplary CEA binding scFv domains are provided in table 38.

Table 38: amino acid sequences of exemplary extracellular CEA binding domains

In one embodiment, the anti-CEA scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:267 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:268 or conservative amino acid substitutions thereof. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has a V with at least 98% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has at least 97% identity V _H And V _L A zone. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has a V with at least 96% identity to the sequence shown at 268 _H And V _L A zone. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has a V with at least 95% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has a V with at least 90% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has a V with at least 85% identity to the sequence shown in SEQ ID NO. 268 _H And V _L A zone. In one embodiment, the anti-CEA scFv domain comprises a sequence that is each of SEQ ID NO:267 and SEQ ID NO:268 has a V with at least 80% identity to the sequence shown in SEQ ID NO. 268 _H And V _L A zone.

In one embodiment, the anti-CEA scFv domain comprises a polypeptide having the sequence of SEQ ID NO: 269. SEQ ID NO:270 and/or SEQ ID NO:271 or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 272. SEQ ID NO:273 and/or SEQ ID NO:274 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown.

In one embodiment, the anti-CEA binding domain comprises scFv, V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a variant, fragment or derivative thereof, or a conservative amino acid substitution thereof, or a nucleotide encoding such a sequence, as disclosed in international patent application publication No. WO 2020/152451 A1, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-CEA binding domain comprises scFv, V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a variant, fragment or derivative thereof, or a conservative amino acid substitution thereof, or a nucleotide encoding such a sequence, as disclosed in U.S. patent application publication No. US 2009/017108 A1, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-CEA binding domain comprises scFv, V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a variant, fragment or derivative thereof, or a conservative amino acid substitution thereof, or a nucleotide encoding such a sequence, as disclosed in U.S. patent No. 5,081,235, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-CEA binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, or variants, fragments, or derivatives thereof, or conservative amino acid substitutions thereof, as disclosed in U.S. patent No. 10,865,243, the disclosure of which is incorporated herein by reference in its entirety.

4. Extracellular CD73 binding domain

In one embodiment, the CCR of the present invention comprises an extracellular domain comprising a CD73 binding domain, also referred to herein as an anti-CD 73 domain. CD73 (also known as exo-5 '-nucleotidase or exo-5' nt) is a glycosyl-phosphatidylinositol (GPI) -linked cell surface enzyme expressed in endothelial cells and hematopoietic cell subsets. Resta et al, immunol. Rev.1998,161,95-109. CD73 is known to catalyze the dephosphorylation of extracellular nucleoside monophosphates to nucleosides, such as adenosine, which are shown to regulate proliferation and migration of many cancers and to have immunosuppressive effects by modulating anti-tumor T cells. Zhang et al, cancer res.2010,70,6407-11). CD73 is expressed in many different cancers, including colon cancer, lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, leukemia, glioma, glioblastoma, melanoma, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer. Jin et al, cancer Res.2010,70,2245-55; stagg et al, proc.Nat' l.Acad.Sci.2010,107,1547-52. Furthermore, CD73 expression in cancer has been linked to increased proliferation, migration, angiogenesis, invasion , metastasis and shorter patient survival. In one embodiment, the CD73 binding domain is an scFv domain. In one embodiment, the CD73 binding domain binds to murine CD 73. In one embodiment, the CD73 binding domain binds to human CD 73. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-CD 73V _H And V _L Domains, linkers are as described herein.

In one embodiment, the anti-CD 73 binding domain comprises V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a nucleotide encoding such a sequence, such as U.S. patent No. 10,287,362; 10,556,968; and 10,864,269, the disclosures of which are incorporated herein by reference in their entirety. The amino acid sequences of exemplary CD73 binding scFv domains are provided in table 39.

Table 39: amino acid sequence of exemplary extracellular CD73 binding domain

In one embodiment, the anti-CD 73 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:275 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:276 or a conservative amino acid substitution thereof. In one embodiment, the anti-CD 73 scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73 scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:275 and SEQ ID NO:276 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-CD 73scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 277. SEQ ID NO:278 and/or SEQ ID NO:279, or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 280. SEQ ID NO:281 and/or SEQ ID NO:282 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequence depicted.

In one embodiment, the anti-CD 73 binding domain comprises V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, as disclosed in U.S. patent No. 9,388,249; the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of additional exemplary CD73 binding scFv domains are provided in table 40.

Table 40: amino acid sequence of exemplary extracellular CD73 binding domain

In one embodiment, the anti-CD 73 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:283 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:284 or conservative amino acid substitutions thereof. In one embodiment, the anti-CD 73 scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73 scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73 scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-CD 73scFv domain comprises a sequence that is each of SEQ ID NO:283 and SEQ ID NO:284 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-CD 73 binding domain comprises V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a conservative amino acid substitution thereof, or a nucleotide encoding such a sequence, as disclosed in U.S. patent No. 10,822,426, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-CD 73 binding domain comprises V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a conservative amino acid substitution thereof, or a nucleotide encoding such a sequence, as disclosed in U.S. patent application publication No. US 2019/0284293 A1, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-CD 73 binding domain comprises V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, or conservative amino acid substitutions, or nucleotides encoding such sequences, e.g., U.S. patent application publication No. US 2020/0392243 A1, the disclosure of which is incorporated herein by reference in its entirety.

5. Extracellular TROP-2 binding domains

In one embodiment, the CCR comprises an extracellular domain, which is a domain capable of binding to human TROP-2. In one embodiment, the extracellular domain binds to human TROP-2 (also known as trophoblast cell surface antigen-2, tumor-associated calcium signal transducer-2, or epithelial glycoprotein-1 antigen (EGP-1)), which is encoded by TACSTD 2. The function of TROP-2 and its role in the pathogenesis of tumors, including its activation of the ERK-MAPK pathway and the PI3K-AKT pathway, is described in Cubas et al, mol. Cancer 2010,9,253; gu et al, mol. Med. Rep.2018,18,1782-88; and mcdougal et al, dev. Dyn.2015,244,99-109, the disclosures of each of which are incorporated herein by reference in their entirety. In one embodiment, the extracellular domain binds to murine or human TROP-2. In one embodiment, the extracellular TROP-2 binding domain is an scFv domain. In one embodiment, the TROP-2 scFv binding domain binds to murine TROP-2. In one embodiment, the TROP-2 scFv binding domain binds to human TROP-2. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-TROP-2V _H And V _L Domains, linkers are as described herein.

In one embodiment, the CCR comprises an extracellular scFv domain that binds to TROP-2 and comprises V _H 、V _L Or CDR domains, or nucleotides encoding such domains, are described in U.S. patent application publication No. US 2012/0237218 A1, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary TROP-2 binding scFv domains are provided in table 41.

Table 41: amino acid sequence of exemplary TROP-2 binding scFv Domain

In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises a sequence selected from SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO: 295. SEQ ID NO:296 and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 297. SEQ ID NO: 298. SEQ ID NO: 299. SEQ ID NO:300 and conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has 99% identity V _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has at least 99% identity V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has a V with 98% identity to the sequence of 296 _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has a V with at least 98% identity to the sequence of 300 _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has 97% identity V _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has at least 97% identity V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has 96% identity V _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has a V with at least 96% identity to the sequence of 300 _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has 95% identity V _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has a V with at least 95% identity to the sequence of 300 _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has a sequence of 90% identity V _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has a V with at least 90% identity to the sequence of 300 _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has 85% identity V _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has a V with at least 85% identity to the sequence of 300 _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 291. SEQ ID NO: 292. SEQ ID NO: 293. SEQ ID NO: 294. SEQ ID NO:295 and SEQ ID NO:296 has a sequence of 80% identity V _H A region and a sequence selected from SEQ ID NO: 297. SEQ ID NO: 298. SEQ ID NO:299 and SEQ ID NO:300 has a V with at least 80% identity to the sequence of 300 _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 301. SEQ ID NO:302 and/or SEQ ID NO:303, or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 304. SEQ ID NO:305 and/or SEQ ID NO:306 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown.

In one embodiment, the CCR comprises an extracellular scFv domain that binds to TROP-2 and comprises a sequence according to SEQ ID NO: v of 292 _H A chain. In one embodiment, the CCR comprises an extracellular scFv domain comprising a sequence according to SEQ ID NO: 298V _H A chain in which at least one amino acid is modified to replace Ala at position 9 with Pro, lys at position 12 with Val, val at position 20 with Ile, arg at position 38 with Lys, met at position 48 with Ile, and position 67 with LysArg, val at position 68 with Ala, ILe at position 70 with Leu, tyr at position 95 with Phe or Val at position 112 with Leu are introduced as the amino acid sequence of SEQ ID NO:292, and a sequence of amino acids thereof. In one embodiment, the extracellular domain is an scFv domain. In one embodiment, the CCR comprises an extracellular scFv domain that binds to TROP-2 and comprises a sequence according to SEQ ID NO: 298V _L A chain.

In one embodiment, the anti-TROP-2 binding domain comprises V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent No. 9,399,074, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the anti-TROP-2 binding domain comprises the V of antibodies m7E6, h7E6, h7E6_SVG, h7E6_SVGL, m6G11, h6G11 or h6G11-FKGSF _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a nucleotide encoding such a sequence, as disclosed in U.S. patent No. 9,399,074, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the anti-TROP-2 binding domain comprises the antibody m7E6, h7E6_SVG4, h7E6_SVG19, h7E6_SVG6, h7E6_SVG20, h7E6_SVG22, h7E6_SVG28, h7E6_SVG30, h7E6_SVGL, h7E6_SVGL1, h7E6_SVGL2, h7E6_SVGL3, h7E6_SVGL4, h7E6_SVGL5, h7E6_SVGN, m6G11, h6G11, or h6G11_FKG_SF V _H And/or V _L A sequence, or a heavy and/or light chain CDR1, CDR2 and/or CDR3 sequence, or a nucleotide encoding such a sequence, as disclosed in U.S. patent No. 9,399,074, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary TROP-2 binding scFv domains are provided in table 42.

Table 42: amino acid sequence of exemplary TROP-2 binding scFv Domain

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In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain or protection thereofConservative amino acid substitutions. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:307 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:308 or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 99% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 98% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 97% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 96% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 95% identity to the sequence shown in 308 _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 90% identity to the sequence shown in 308 _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 85% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 307 and SEQ ID NO:308 has a V with at least 80% identity to the sequence shown in seq id no _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain or conservative amino acid substitutions thereof. At the position ofIn one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:309 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:310 or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310, and V having at least 99% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310 has a V with at least 98% identity to the sequence shown in 310 _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310, and V having at least 97% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310, and a V having at least 96% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310, and V having at least 95% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310, and a V having at least 90% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310, and a V having at least 85% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 309 and SEQ ID NO:310, and V having at least 80% identity to the sequence set forth in seq id no _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain or conservative amino acid substitutions thereof. In one embodiment of the present invention, in one embodiment,the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:311 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:310 or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310, and V having at least 99% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310 has a V with at least 98% identity to the sequence shown in 310 _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310, and V having at least 97% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310, and a V having at least 96% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310, and V having at least 95% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310, and a V having at least 90% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310, and a V having at least 85% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:310, and V having at least 80% identity to the sequence set forth in seq id no _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv structureDomain contains V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:311 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:312 or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 99% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 98% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 97% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 96% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 95% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 90% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 85% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 311 and SEQ ID NO:312 has a V with at least 80% identity to the sequence shown in seq id no _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:313 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:314 or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 99% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 98% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 97% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 96% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 95% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 90% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 85% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 313 and SEQ ID NO:314 has a V with at least 80% identity to the sequence shown in seq id no _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:315 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:316 or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has at least 99% identity V to the sequence shown _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has a V with at least 98% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has at least 97% identity V to the sequence shown _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has a V with at least 96% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has a V with at least 95% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has a V with at least 90% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has a V with at least 85% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 315 and SEQ ID NO:316 has a V with at least 80% identity to the sequence shown in seq id no _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:317 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:318 or a conservative amino acid substitution thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 99% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 98% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 97% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 96% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 95% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 90% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 85% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 317 and SEQ ID NO:318 has a V with at least 80% identity to the sequence shown in seq id no _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 319. SEQ ID NO:320 and/or SEQ ID NO:321, and/or has a heavy chain CDR1, CDR2, and CDR3 domain of the sequence set forth in SEQ ID NO: 322. SEQ ID NO:323 and/or SEQ ID NO:324 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown.

Exemplary TROP-2 binding V of scFv domains encoding m7E6, h7E6, h7E6_SVG, h7E6_SVGL, m6G11, h6G11 and h6G11-FKG_SF _H And V _L The nucleotide sequences of the domains are provided in table 43 and further described in U.S. patent No. 9,399,074, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the nucleotide sequences in table 43 are codon optimized to improve protein expression.

Table 43: nucleotide sequence of exemplary TROP-2 binding scFv Domain

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In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:325, light chain variable region (V _L ) Consists of SEQ ID NO: 326. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326 has a nucleotide encoding V having at least 99% identity to the sequence depicted in figure 1 _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326 has a nucleotide encoding V having at least 98% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326 has a nucleotide encoding V having at least 97% identity to the sequence depicted in figure 1 _H And/or V _L A zone. In one embodimentIn, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326 has a nucleotide-encoded V with at least 96% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326 has a nucleotide encoding V having at least 95% identity to the sequence depicted in figure 1 _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326 has a nucleotide encoding V having at least 90% identity to the sequence depicted in figure 1 _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326 has a nucleotide encoding V having at least 85% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:325 and SEQ ID NO:326, and a V encoded by a nucleotide having at least 80% identity to the sequence depicted in seq id no _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:325 and/or SEQ ID NO:326 is codon optimized to improve protein expression.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:327, a light chain variable region (V _L ) Consists of SEQ ID NO: 328. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 99% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 98% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one implementationIn a mode, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 97% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 96% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 95% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 90% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 85% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:327 and SEQ ID NO:328 has a nucleotide encoding V having at least 80% identity to the sequence depicted in 328 _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:327 and/or SEQ ID NO:328 codon optimized to improve protein expression.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:329, a light chain variable region (V _L ) Consists of SEQ ID NO: 328. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 99% identity to the sequence depicted in seq id no _H And/or V _L A zone. At the position ofIn one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 98% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 97% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 96% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 95% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 90% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 85% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:328 has a nucleotide encoding V having at least 80% identity to the sequence depicted in seq id no _H And/or V _L A zone. In embodiments including the foregoing embodiments, the nucleotide sequences in table 43 are codon optimized to improve protein expression. In embodiments including the preceding embodiments, SEQ ID NO:329 and/or SEQ ID NO:328 codon optimized to improve protein expression.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:329, a light chain variable region (V _L ) Consists of SEQ ID NO: 330. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 99% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 98% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 97% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 96% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 95% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 90% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 85% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:329 and SEQ ID NO:330, and a V encoded by a nucleotide having at least 80% identity to the sequence set forth in seq id no _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:329 and/or SEQ ID NO:330 was codon optimized to improve protein expression.

In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide encoded by a nucleotide sequenceHeavy chain variable region (V) of scFv antibody m6G11 _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:331, the light chain variable region (V _L ) Consists of SEQ ID NO: 332. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 99% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 98% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 97% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 96% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 95% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 90% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 85% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:331 and SEQ ID NO:332, and a V encoded by a nucleotide having at least 80% identity to the sequence set forth in seq id no _H And/or V _L A zone. In a case of including the foregoing embodimentIn embodiments, SEQ ID NO:331 and/or SEQ ID NO:332 codon optimized to improve protein expression.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:333, the light chain variable region (V _L ) Consists of SEQ ID NO: 334. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 99% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 98% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 97% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 96% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 95% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 90% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 85% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodimentWherein the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:333 and SEQ ID NO:334, and a V encoded by a nucleotide having at least 80% identity to the sequence shown in seq id no _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:333 and/or SEQ ID NO:334 was codon optimized to improve protein expression.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:335, a light chain variable region (V _L ) Consists of SEQ ID NO: 336. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 99% identity to a nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 98% identity to a nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 97% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 96% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 95% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 90% identity to the nucleotide-encoded V _H And/or V _L A zone. At the position ofIn one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 85% identity to a nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide consisting of a sequence that is separated from SEQ ID NO:335 and SEQ ID NO:336 having at least 80% identity to a nucleotide-encoded V _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:335 and/or SEQ ID NO:336 are codon optimized to improve protein expression.

In one embodiment, the CCR comprises an extracellular scFv domain that binds to TROP-2 and comprises V of Sha Xituo bead mab _H 、V _L Or CDR domains, or fragments, derivatives, or variants thereof. Preparation and Properties of Sha Xituo bead mab (an anti-TROP-2 monoclonal antibody) and V thereof _H 、V _L CDRs and other related domains, including amino acid and nucleotide sequences thereof, are described in U.S. patent No. 9,770,517, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary TROP-2 binding domains for CCR of the present invention are provided in table 44.

Table 44: sha Xituo amino acid sequence of the TROP-2-binding scFv Domain of the bead monoclonal antibody

In one embodiment, the anti-TROP-2 scFv domain comprises V of Sha Xituo bead mab _H Domain and/or V _L A domain, or a fragment, variant, derivative or biological analogue thereof. In one embodiment, the anti-TROP-2 scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain comprises SEQ ID NO:337 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:338 or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 as shown in the figureV having at least 99% identity to the sequence _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 has a V with at least 98% identity to the sequence shown in fig _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 has a V with at least 97% identity to the sequence shown in fig _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 has a V with at least 96% identity to the sequence shown in fig _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 has a V with at least 95% identity to the sequence shown in fig _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 has a V with at least 90% identity to the sequence shown in fig _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 has a V with at least 85% identity to the sequence shown in fig _H And/or V _L A zone. In one embodiment, the anti-TROP-2 scFv domain comprises a sequence that is complementary to each of SEQ ID NOs: 337 and/or SEQ ID NO:338 has a V with at least 80% identity to the sequence shown in fig _H And/or V _L A zone.

In one embodiment, the anti-TROP-2 scFv domain comprises the heavy chain CDR1, CDR2, and/or CDR3 domains of Sha Xituo bead mab or conservative amino acid substitutions thereof and/or the light chain CDR1, CDR2, and CDR3 domains of sabitumomab or conservative amino acid substitutions thereof. In one embodiment, the anti-TROP-2 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 339. SEQ ID NO:340 and/or SEQ ID NO:341, or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 342. SEQ ID NO:343 and/or SEQ ID NO:344 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown.

In one embodiment, the CCR comprises an extracellular scFv domain or V _H And/or V _L Or heavy and/or light chain CDR1, CDR2 and/or CDR3 domains which bind to TROP-2 and are disclosed in U.S. patent No. 9,062,100; 9,670,287; 9,850,312; 10,202,461; and U.S. patent application publication No. US 2019/0144559 A1, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the CCR comprises an extracellular scFv domain or V _H And/or V _L Or heavy and/or light chain CDR1, CDR2 and/or CDR3 domains, which are combined with TROP-2 from antibody ar47a6.4.2 disclosed in U.S. patent application publication No. US 2008/013428 A1, the disclosure of which is incorporated herein by reference in its entirety.

6. Extracellular EPCAM binding domains

In one embodiment, the CCR comprises an extracellular domain, which is a domain capable of binding EPCAM, also known as an epithelial cell adhesion molecule, tumor associated calcium signal transducer 1 or tactd 1, CD326 and 17-A1 antigen. The domain capable of binding to EPCAM is also referred to herein as an anti-EPCAM domain. In one embodiment, the extracellular domain binds to human EPCAM. In one embodiment, the extracellular domain binds to murine EPCAM. In one embodiment, the extracellular EPCAM binding domain is an scFv domain that binds to human EPCAM or murine EPCAM. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-EPCAM V _H And V _L Domains, linkers are as described herein.

In some embodiments, the EPCAM binding domains include the scFv domains of antibodies 3-17I scFv, 7-F17 scFv, 12-C15 scFv, 16-G5 scFv, 17-C20 scFv, and 24-G6 scFv, as well as fragments, variants, and derivatives thereof, each as described in U.S. Pat. No. 8,637,017, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary EPCAM binding domains for CCR of the present invention are provided in table 45.

Table 45: amino acid sequence of exemplary EPCAM binding scFv domains

In one embodiment, the anti-EPCAM scFv domain comprises the amino acid sequence of SEQ ID NO:345 or conservative amino acid substitutions thereof. In one embodiment, the anti-EPCAM scFv domain comprises a scFv antibody 3-17I scFv or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 99% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 98% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 97% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 96% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 95% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 90% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 85% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:345 has an scFv domain with at least 80% identity.

In one embodiment, the anti-EPCAM scFv domain comprises the amino acid sequence of SEQ ID NO:346 or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises the scFv antibody 7-F17 scFv or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346 has an scFv domain with at least 99% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346 has an scFv domain with at least 98% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346 has an scFv domain with at least 97% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346 has an scFv domain with at least 96% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346 has an scFv domain with at least 95% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346 has an scFv domain with at least 90% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346 has an scFv domain with at least 85% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:346, has an scFv domain with at least 80% identity.

In one embodiment, the anti-EPCAM scFv domain comprises the amino acid sequence of SEQ ID NO:347 or conservative amino acid substitutions thereof. In one embodiment, the anti-EPCAM scFv domain comprises scFv antibody 12-C15 scFv or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain with at least 99% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain with at least 98% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain with at least 97% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain with at least 96% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain having at least 95% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain having at least 90% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain having at least 85% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:347 has an scFv domain having at least 80% identity.

In one embodiment, the anti-EPCAM scFv domain comprises the amino acid sequence of SEQ ID NO:348 or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises the scFv antibody 16-G5 scFv or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain having at least 99% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain having at least 98% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain having at least 97% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain with at least 96% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain with at least 95% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain having at least 90% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain having at least 85% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:348 has an scFv domain having at least 80% identity.

In one embodiment, the anti-EPCAM scFv domain comprises the amino acid sequence of SEQ ID NO:349 or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises scFv antibody 17-C20 scFv or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 99% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 98% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 97% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 96% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 95% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 90% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 85% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:349 has an scFv domain having at least 80% identity.

In one embodiment, the anti-EPCAM scFv domain comprises the amino acid sequence of SEQ ID NO:350 or conservative amino acid substitutions thereof. In one embodiment, the anti-EPCAM scFv domain comprises the scFv antibody 24-G6 scFv or a conservative amino acid substitution thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 99% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 98% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 97% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 96% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 95% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 90% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 85% identity. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that hybridizes to SEQ ID NO:350 has an scFv domain with at least 80% identity.

In one embodiment, the anti-EPCAM scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises a sequence selected from SEQ ID NOs: 351 and fragments, derivatives, variants and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives, variants, and conservative amino acid substitutions thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have 99% identity V _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 99% identity in sequence _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have a V with 98% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 98% identity V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have 97% identity V _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 98% identity V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have 96% identity V _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 96% identity V _L A zone. In a real worldIn embodiments, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have 95% identity V _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 95% identity in sequence _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have a V with 90% identity to the sequence of the fragment _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 90% identity V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have a V sequence of 85% identity _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 85% identity V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 351 and fragments, derivatives and variants thereof, have a sequence of 80% identity V _H A region and a sequence selected from SEQ ID NO: 352. SEQ ID NO: 353. SEQ ID NO: 354. SEQ ID NO: 355. SEQ ID NO: 356. SEQ ID NO:357 and fragments, derivatives and variants thereof, and have at least 80% identity V _L A zone.

In one embodiment, the anti-EPCAM scFv domain comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 358. SEQ ID NO:359 and/or SEQ ID NO:360, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 361. SEQ ID NO:362 and/or SEQ ID NO:363 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown.

Nucleotide sequences encoding exemplary EPCAM binding scFv domains of antibodies 3-17I scFv, 7-F17 scFv, 12-C15 scFv, 16-G5 scFv, 17-C20 scFv and 24-G6 scFvFragments, variants and derivatives and V encoding additional scFv domains _H And V _L The nucleotide sequence of the domains is provided in table 46 and further described in U.S. patent No. 8,637,017, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the nucleotide sequences in table 46 are codon optimized to improve protein expression.

Table 46: nucleotide sequence of exemplary EPCAM binding scFv domains

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In one embodiment, the anti-EPCAM scFv domain comprises a scFv antibody 3-17I scFv encoded by a nucleotide sequence. In one embodiment, the anti-EPCAM scFv domain consists of SEQ ID NO: 364. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding at least 99% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding at least 98% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding at least 97% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding at least 96% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding at least 95% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding at least 90% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding a sequence having at least 85% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:364, a nucleotide sequence encoding at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:364 are codon optimized to improve protein expression.

In one embodiment, the anti-EPCAM scFv domain comprises the scFv antibody 7-F17 scFv encoded by a nucleotide sequence. In one embodiment, the anti-EPCAM scFv domain consists of SEQ ID NO: 365. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 99% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 98% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 97% identity thereto. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 96% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 95% identity thereto. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 90% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 85% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:365, and a nucleotide sequence encoding at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:365 codon optimized to improve protein expression.

In one embodiment, the anti-EPCAM scFv domain comprises a scFv antibody 12-C15 scFv encoded by a nucleotide sequence. In one embodiment, the anti-EPCAM scFv domain consists of SEQ ID NO:366, and a sequence code as shown in seq id no. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 99% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 98% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 97% identity to the nucleotide sequence encoding the sequence set forth in seq id no. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 96% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 95% identity to the nucleotide sequence encoding the sequence set forth in seq id no. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 90% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 85% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:366 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:366 was codon optimized to improve protein expression.

In one embodiment, the anti-EPCAM scFv domain comprises a scFv antibody 16-G5 scFv encoded by a nucleotide sequence. In one embodiment, the anti-EPCAM scFv domain consists of SEQ ID NO: 367. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence encoding a sequence having at least 99% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence encoding a sequence having at least 98% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence having at least 97% identity thereto. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence encoding a sequence having at least 96% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence having at least 95% identity thereto. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence having at least 90% identity thereto. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence encoding a sequence having at least 85% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:367, a nucleotide sequence having at least 80% identity thereto. In embodiments including the preceding embodiments, SEQ ID NO:367 is codon optimized to improve protein expression.

In one embodiment, the anti-EPCAM scFv domain comprises a scFv antibody 17-C20 scFv encoded by a nucleotide sequence. In one embodiment, the anti-EPCAM scFv domain consists of SEQ ID NO: 368. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 99% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 98% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 97% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 96% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 95% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 90% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 85% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:368, a nucleotide sequence encoding a sequence having at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:368 is codon optimized to improve protein expression.

In one embodiment, the anti-EPCAM scFv domain comprises a scFv antibody 24-G6 scFv encoded by a nucleotide sequence. In one embodiment, the anti-EPCAM scFv domain consists of SEQ ID NO: 369. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding a sequence having at least 99% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding a sequence having at least 98% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding a sequence having at least 97% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding at least 96% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding a sequence having at least 95% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding a sequence having at least 90% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding a sequence having at least 85% identity. In one embodiment, the anti-EPCAM scFv domain consists of a sequence that hybridizes to SEQ ID NO:369, a nucleotide sequence encoding a sequence having at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:369 is codon optimized to improve protein expression.

In one embodiment, the anti-EPCAM scFv domain comprises V _H Domain and V _L Domain, V _H The domain consists of a sequence selected from SEQ ID NOs: 370 and fragments, derivatives, variants and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 371. SEQ ID NO: 372. SEQ ID NO: 372. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives, variants and conservative amino acid substitutions thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereof, and V having 99% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, and V having at least 99% identity to the sequence of the variant _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereof, and V with 98% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, and V having at least 98% identity to the sequence of the variant _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereof, and V having 97% identity to the sequence _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, and V having at least 97% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereof, have 96% identity V _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, have a V of at least 96% identity _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereof, and V with 95% identity to the sequence _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, and V having at least 95% identity to the sequence of the variant _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereof, and V having 90% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, and V having at least 90% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereofV with 85% identity _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, and V having at least 85% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 370 and fragments, derivatives and variants thereof, and V having 80% identity to the sequence _H A region and a sequence selected from SEQ ID NO: 371. SEQ ID NO: 372. SEQ ID NO: 373. SEQ ID NO: 374. SEQ ID NO: 375. SEQ ID NO:376 and fragments, derivatives and variants thereof, and V having at least 80% identity to the sequence of the variant _L A zone.

In some embodiments, EPCAM binding domains include scFv, VH, VL and CDR domains as described in U.S. patent No. 9,388,249, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary EPCAM binding domains for CCR of the present invention are provided in table 47.

Table 47: amino acid sequence of exemplary extracellular EPCAM binding domains

In one embodiment, the anti-EPCAM scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:377 or conservative amino acid substitutions, light chain variable region (V _L ) Comprising SEQ ID NO:378 or conservative amino acid substitutions thereof. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 99% identity _H And V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 98% identity _H And V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 97% identity _H And V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 96% identity _H And V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 90% identity thereto _H And V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-EPCAM scFv domain comprises a sequence that is each of SEQ ID NO:377 and SEQ ID NO:378 has a V with at least 80% identity _H And V _L A zone.

In one embodiment, the anti-EPCAM scFv domain comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 379. SEQ ID NO:380 and/or SEQ ID NO:381, or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 382. SEQ ID NO:383 and/or SEQ ID NO:384 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-EPCAM binding domain comprises an additional scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent No. 9,388,249, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-EPCAM binding domain comprises an additional scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent application publication No. US 2019/023536 A1, the disclosure of which is incorporated herein by reference in its entirety.

7. Extracellular tissue factor binding domains

In one embodiment, a CCR of the present invention comprises an extracellular domain comprising a Tissue Factor (TF) binding domain, also referred to herein as an anti-TF domain. TF is a transmembrane glycoprotein having 219 amino acid residue extracellular regions, 23 amino acid residue transmembrane regions, and 21 amino acid residue cytoplasmic regions, which in combination with factor VIIa initiate blood clotting. TF is expressed in lung, pancreas, breast, colon and stomach cancers. Hu et al, oncol. Res.1994,6,321-327; callander et al, cancer 1992,70,1194-201. Abnormally high expression of TF has been clinically shown to be associated with poor differentiation of many tumors, including colorectal, NSCLC and breast cancers. Shigernori et al, thromb.Haemost.1998,80,894-898; seto et al, cancer 2000,88,295-301; sawada et al, br.J.cancer 1999,79,472-477; kirschmann et al, breast Cancer Res. Treat.1999,55,127-136; schwirzke et al, statics Res.1999,19,1801-1814. In one embodiment, the TF binding domain is an scFv domain. In one embodiment, the CCR comprises an extracellular domain that binds to human TF. In one embodiment, the extracellular domain binds to murine TF. In one embodiment, the extracellular TF binding domain is an scFv domain. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-TF V _H And V _L Domains, linkers are as described herein.

In one embodiment, the TF binding domain comprises the CDRs, V as described in U.S. patent No. 7,993,644 _H And V _L Domain-prepared scFv antibodies, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the scFv domain comprises scFv, V, of antibody TF260, TF196, TF278, TF277, TF392 or TF9 _H 、V _L Or CDR domains, their respective preparations and properties are described in U.S. patent No. 7,993,644 and incorporated by reference in its entirety, including V for each of TF260, TF196, TF278, TF277, TF392 or TF9 _H 、V _L And CDR domains. In one embodiment, the scFv comprises a Talcum Shu Tushan anti-scFv, V _H 、V _L Or a CDR domain, or variant, fragment or derivative thereof, the structure of which, together with the availability ofOther scFv, V in embodiments of the invention _H 、V _L Or CDR domains are described in U.S. patent application publications US 2019/0169311 A1, US 2019/0315880 A1, US 2020/024677 A1 and US 2021/0030888 A1, the disclosures of which are incorporated herein by reference in their entirety. In one embodiment, the scFv comprises an antibody described in U.S. patent No. 9,168,314, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the scFv comprises an antibody described in U.S. patent No. 7,824,677, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary TF-binding scFv domains are provided in table 48.

Table 48: amino acid sequence of exemplary tissue factor binding scFv domains

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In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:385 or conservative amino acid substitutions, light chain variable region (V _L ) Comprising SEQ id no:386 or conservative amino acid substitutions thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:385 and SEQ ID NO:386 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:385 and SEQ ID NO:386 has a V with at least 98% identity to the sequence shown in 386 _H And V _L A zone. In one embodiment, the anti-TF scFv domains comprise eachAnd (3) respectively comparing with SEQ ID NO:385 and SEQ ID NO:386 has at least 97% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:385 and SEQ ID NO:386 has a V with at least 96% identity to the sequence shown in 386 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:385 and SEQ ID NO:386 has at least 95% identity to the sequence shown in V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:385 and SEQ ID NO:386 has at least 90% identity to the sequence shown in V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:385 and SEQ ID NO:386 has a V with at least 85% identity to the sequence shown in 386 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:385 and SEQ ID NO:386 has at least 80% identity to the sequence shown in V _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:387 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:388 or conservative amino acid substitutions thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has at least 99% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has at least 98% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has a sequence of at least 9V of 7% identity _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has at least 96% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has at least 95% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has at least 90% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has at least 85% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:387 and SEQ ID NO:388 has at least 80% identity of V _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:389 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:390 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodimentIn (c), the anti-TF scFv domain comprises a sequence that is distinguishable from SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:389 and SEQ ID NO:390 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:391 or conservative amino acid substitutions, light chain variable region (V _L ) Comprising SEQ ID NO:392 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:391 and SEQ ID NO:392 has at least 99% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:391 and SEQ ID NO:392 has at least 98% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:391 and SEQ ID NO:392 has at least 97% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ id no:391 and SEQ IDNO:392 has at least 96% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:391 and SEQ ID NO:392 has at least 95% identity of V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:391 and SEQ ID NO:392 has at least 90% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:391 and SEQ ID NO:392 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:391 and SEQ ID NO:392 has at least 80% identity of V _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:393 or conservative amino acid substitutions, light chain variable region (V _L ) Comprising SEQ ID NO:394 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has at least 99% identity to V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has a V with at least 98% identity to the sequence shown in 394 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has at least 97% identity to the sequence shown in V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has at least 96% identity V to the sequence shown in 394 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has at least 95% identity to V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has at least 90% identity to the sequence shown in V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has a V with at least 85% identity to the sequence shown in 394 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:393 and SEQ ID NO:394 has at least 80% identity to V _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:395 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:396 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:395 and SEQ ID NO:396 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:395 and SEQ ID NO:396 has at least 98% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:395 and SEQ ID NO:396 has at least 97% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:395 and SEQ ID NO:396, having at least 96% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domains compriseSelf-discrimination with SEQ ID NO:395 and SEQ ID NO:396, having at least 95% identity to V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:395 and SEQ ID NO:396, having at least 90% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:395 and SEQ ID NO:396, having at least 85% identity V _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:395 and SEQ ID NO:396, having at least 80% identity to V _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises a sequence selected from SEQ ID NOs: 385. SEQ ID NO: 387. SEQ ID NO: 389. SEQ ID NO: 391. SEQ ID NO: 393. SEQ ID NO:395 and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 386. SEQ ID NO: 388. SEQ ID NO: 390. SEQ ID NO: 392. SEQ ID NO: 394. SEQ ID NO:396 and conservative amino acid substitutions thereof.

In one embodiment, the anti-TF scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 397. SEQ ID NO:398 and/or SEQ ID NO:399 or conservative amino acid substitutions thereof, and/or having heavy chain CDR1, CDR2, and CDR3 domains of the sequences set forth in SEQ ID NOs: 400. SEQ ID NO:401 and/or SEQ ID NO:402, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-TF scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 403. SEQ ID NO:404 and/or SEQ ID NO:405 or conservative amino acid substitutions thereof, and/or having heavy chain CDR1, CDR2, and CDR3 domains of the sequences set forth in SEQ ID NOs: 406. SEQ ID NO:407 and/or SEQ ID NO:408 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequence depicted.

In one embodiment, the anti-TF scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 409. SEQ ID NO:410 and/or SEQ ID NO:411, and/or have conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 412. SEQ ID NO:413 and/or SEQ ID NO:414 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-TF scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:415 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:416 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:415 and SEQ ID NO:416 has the sequence shown inV of at least 80% identity _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:417 or a conservative amino acid substitution thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:418 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418, and a V having at least 96% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418, and a V having at least 90% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:417 and SEQ ID NO:418, and a V having at least 80% identity to the sequence set forth in seq id no _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:419 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:420 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420, and V having at least 99% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420, and V having at least 96% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420, and V having at least 95% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420, and V having at least 90% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420, and V having at least 85% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:419 and SEQ ID NO:420, and V having at least 80% identity to the sequence set forth in seq id no _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:421 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:422 or conservative amino acid substitutions thereof. In one embodiment, the anti-TF scFv domain comprises a polypeptide that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 95% identity to the sequence shown in figure 422 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:421 and SEQ ID NO:422 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-TF scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises a sequence selected from SEQ ID NOs: 415. SEQ ID NO: 417. SEQ ID NO: 419. SEQ ID NO:421 and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 416. SEQ ID NO: 418. SEQ ID NO: 420. SEQ ID NO:422 and conservative amino acid substitutions thereof.

In one embodiment, the anti-TF scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:423 or a conservative amino group thereofAcid substitution, light chain variable region (V _L ) Comprising SEQ ID NO:424 or a conservative amino acid substitution thereof. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 99% identity to the sequence shown at 424 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 98% identity to the sequence shown at 424 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 97% identity to the sequence shown at 424 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 96% identity to the sequence shown at 424 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 95% identity to the sequence shown at 424 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 90% identity to the sequence shown at 424 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 85% identity to the sequence shown at 424 _H And V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence that is each of SEQ ID NO:423 and SEQ ID NO:424 has a V with at least 80% identity to the sequence shown at 424 _H And V _L A zone.

Exemplary TF binding V of scFv domains encoding TF260, TF196, TF278, TF277, TF392 and TF9 _H And V _L The nucleotide sequence of the domains is provided in table 49 and further described in U.S. patent No. 7,993,644, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the nucleotide sequences in table 49 are codon optimized to improve protein expression.

Table 49: nucleotide sequence of exemplary tissue factor binding Single chain antibody Domain

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TF scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:425, the light chain variable region (V _L ) Consists of SEQ ID NO: 426. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 99% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 98% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 97% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 96% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 95% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 90% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 85% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:425 and SEQ ID NO:426 has at least 80% identity to the nucleotide-encoded V _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:425 and/or SEQ ID NO:426 was codon optimized to improve protein expression.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TF scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:427, a light chain variable region (V _L ) Consists of SEQ ID NO: 428. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:427 and SEQ ID NO:428 has a nucleotide encoding V having at least 99% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:427 and SEQ ID NO:428 has a nucleotide encoding V with at least 98% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:427 and SEQ ID NO:428 has a nucleotide encoding V with at least 97% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:427 and SEQ ID NO:428 has a nucleotide encoding V having at least 96% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:427 and SEQ ID NO:428 has a nucleotide encoding V having at least 95% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodimentIn (c), the anti-TF scFv domain comprises a sequence consisting of a sequence corresponding to SEQ ID NO:427 and SEQ ID NO:428 has a nucleotide encoding V having at least 90% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:427 and SEQ ID NO:428 has a nucleotide encoding V having at least 85% identity to the sequence depicted in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:427 and SEQ ID NO:428 has at least 80% identity to the nucleotide-encoded V _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:427 and/or SEQ ID NO:428 are codon optimized to improve protein expression.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TF scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:429 and the light chain variable region (V _L ) Consists of SEQ ID NO: 430. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 99% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 98% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 97% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 96% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domainComprises a sequence represented by SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 95% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 90% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 85% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:429 and SEQ ID NO:430, and a V encoded by a nucleotide having at least 80% identity to the sequence set forth in seq id no _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:429 and/or SEQ ID NO:430 was codon optimized to improve protein expression.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TF scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:431 and the light chain variable region (V _L ) Consists of SEQ ID NO: 432. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 99% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 98% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 97% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment of the present invention, in one embodiment, the anti-TF scFv domain comprises is respectively separated from SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 96% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 95% identity to the sequence set forth in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 90% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 85% identity to the sequence shown in seq id no _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:431 and SEQ ID NO:432, and a V encoded by a nucleotide having at least 80% identity to the sequence shown in seq id no _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:431 and/or SEQ ID NO:432 is codon optimized to improve protein expression.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TF scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:433 and the light chain variable region (V _L ) Consists of SEQ ID NO:434, and the sequence code shown in seq id no. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, and a V encoded by a nucleotide having at least 99% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, and a V encoded by a nucleotide having at least 98% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, a nucleotide encoding V having at least 97% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, a nucleotide encoding V having at least 96% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, and a V encoded by a nucleotide having at least 95% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, and a V encoded by a nucleotide having at least 90% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, a nucleotide encoding V having at least 85% identity to the sequence depicted therein _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:433 and SEQ ID NO:434, and a V encoded by a nucleotide having at least 80% identity to the sequence depicted therein _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:433 and/or SEQ ID NO:434 are codon optimized to improve protein expression.

In one embodiment, the anti-TF scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TF scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:435 and the light chain variable region (V _L ) Consists of SEQ ID NO: 436. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has at least 99% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has the sequence shown in seq id noNucleotide-encoded V of at least 98% identity _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has at least 97% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has at least 96% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has at least 95% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has at least 90% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has a nucleotide encoding V having at least 85% identity to the sequence shown in 436 _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:435 and SEQ ID NO:436 has at least 80% identity to the nucleotide-encoded V _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:435 and/or SEQ ID NO:436 are codon optimized to improve protein expression.

8. Extracellular LFA-1 binding domains

In one embodiment, the CCR of the invention comprises an extracellular domain comprising a domain capable of binding to a T cell integrin known as lymphocyte function associated antigen 1 (LFA-1), also referred to herein as an anti-LFA-1 domain. thealphasubunitofLFA-1isdesignatedCD11aanditsligandsincludeICAM-1,ICAM-2,ICAM-3,ICAM-4,ICAM-5andJAM-A. LFA-1 is described in more detail in Walling and Kim, front. Immunol.,2018,9,1-10, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the extracellular domain binds to human LFA-1. In a real world In embodiments, the extracellular domain binds to murine LFA-1. In one embodiment, the extracellular LFA-1 binding domain is an scFv domain that binds to human LFA-1 or murine LFA-1. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The structural domain is LFA-1V resistant _H And V _L Domains, linkers are as described herein. In one embodiment, the extracellular domain binds to human CD11a (also referred to herein as anti-CD 11 a). In one embodiment, the extracellular domain binds to murine CD11 a. In one embodiment, the extracellular CD11a binding domain is an scFv domain that binds to human CD11a or murine CD11 a. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-CD 11aV _H And V _L Domains, linkers are as described herein.

In some embodiments, the LFA-1 binding domain or CD11a binding domain of a CCR of the invention comprises an scFv domain of an antibody described in U.S. patent application publication No. US 2015/0079075 A1. The amino acid sequences of exemplary LFA-1 or CD11a binding domains for use in CCR of the present invention are provided in table 50.

Table 50: exemplary amino acid sequences of LFA-1 or CD11 a-binding scFv domains

In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:437 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:438 or conservative amino acid substitutions thereof. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprisesEach is isolated from SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a domain comprises a polypeptide that is each of SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:437 and SEQ ID NO:438 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:439 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:440 or conservative amino acid substitutions thereof. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:439 and SEQ ID NO:440, and a V having at least 99% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:439 and SEQ ID NO:440 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodimentWherein the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that hybridizes to SEQ ID NO:439 and SEQ ID NO:440 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:439 and SEQ ID NO:440, and a V having at least 96% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:439 and SEQ ID NO:440, and a V having at least 95% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:439 and SEQ ID NO:440, and the sequence shown has at least 90% identity V _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:439 and SEQ ID NO:440, and a V having at least 85% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a sequence that is each of SEQ ID NO:439 and SEQ ID NO:440, and a V having at least 80% identity to the sequence set forth in seq id no _H And V _L A zone.

In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises a sequence selected from SEQ ID NOs: 437. SEQ ID NO:439 and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 438. SEQ ID NO:440 and conservative amino acid substitutions thereof.

In one embodiment, the anti-LFA-1 or anti-CD 11a scFv domain comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 441. SEQ ID NO:442 and/or SEQ ID NO:443, or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 444. SEQ ID NO:445 and/or SEQ ID NO:446 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown.

In one embodiment, the anti-LFA-1 binding domain or anti-CD 11a binding domain packageOlympic mab scFv, V _H And/or V _L Sequences or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences or derivatives, variants or fragments thereof or humanized variants thereof. Olymomomab is available from Creative Biolabs, inc. (Shirley, N.Y..

In one embodiment, additional scFv, V are included _H And/or V _L The anti-LFA-1 binding domain or anti-CD 11a binding domain of the sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, is an antibody derived from the disclosure of U.S. patent No. 5,284,931, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, additional scFv, V are included _H And/or V _L The anti-LFA-1 binding domains of sequences or heavy chain and/or light chain CDR1, CDR2 and/or CDR3 sequences are obtained from antibodies produced by cell lines deposited in ATCC, including M17/4.4 (ATCC TIB-217), TS2/18.1.1 (ATCC HB-195), TS1/22.1.1.13 (ATCC HB-202), TS1/18.1.2.11 (ATCC HB-203), LM2/1.6.11 (ATCC HB-204), TS2/9.1.4.3 (ATCC HB-205), 2E6 (ATCC HB-226), BE29G1 (ATCC HB-233), TS2/16.2.1 (ATCC HB-243), TS2/4.1.1 (ATCC HB-244), TS2/7.1.1 (ATCC HB-245), S6F1 (ATCC HB-9579), M5/114.15.2 (ATCC IIB-120), M1/70.15.11.5HL (ATCC TIB-128), 441.8 (ATCC HB-213), M17/4.4.11.9 (ATCC TIB-217), M2/7.1.1 (ATCC HB-9579), M2/1.1 (ATCC HB-245), S6F1 (ATCC HB-9579), M5/114.15.2 (ATCC HB-80), M1/C1 (ATCC HB-35.20) and ATCC TIB-48.

In one embodiment, the anti-LFA-1 binding domain or anti-CD 11a binding domain comprises an additional antibody comprising scFv, V as disclosed in U.S. patent application publication No. US 2008/0038259 A1 _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, the disclosures of which are incorporated herein by reference in their entirety.

9. Extracellular FAP binding domains

In one embodiment, the CCR comprises an extracellular domain, which is a domain capable of binding to human FAP. In one embodiment, the extracellular domain binds to human FAP, also known as fibroblast activation protein and fibroblast activation protein alpha. Function of FAP and its application in solid tumors including connective tissueRoles in the parenchyma of the tissue hyperplasia and its expression on Cancer-associated fibroblasts found in breast, lung, colon, pancreatic and other tumors are described in Liu et al, cancer Biol.&Thor.2012, 13,123-129, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the extracellular FAP binding domain is an scFv domain. In one embodiment, the FAP scFv binding domain binds to murine FAP. In one embodiment, the FAP scFv binding domain binds to human FAP. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is FAP V resistant _H And V _L Domains, linkers are as described herein.

In one embodiment, a CCR of the invention comprises an extracellular domain comprising a FAP binding domain. In one embodiment, the FAP binding domain is an anti-FAP binding domain described in U.S. patent application publication No. US 2019/023536 A1, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the anti-FAP binding domain comprises scFv, V of cetrimab (also known as BIBH 1) _H And/or V _L Sequences or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, which are described in U.S. patent application publication No. US 2003/0103968 A1, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the anti-FAP binding domain comprises scFv, V of FAP5 _H And/or V _L Sequences or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, which are described in U.S. patent application publication No. US 2009/0304718 A1, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary FAP-binding scFv domains are provided in table 51.

Table 51: amino acid sequence of exemplary FAP-binding scFv domains

In one embodiment, the anti-FAP scFv domain comprises SEQ ID NO:447 or a conservative amino acid substitution thereof. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 99% identity. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 98% identity. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 97% identity. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 96% identity. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 95% identity. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 90% identity. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 85% identity. In one embodiment, the anti-FAP scFv domain comprises a sequence that hybridizes to SEQ ID NO:447 has an scFv domain with at least 80% identity.

In one embodiment, the anti-FAP scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:448 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:449 or a conservative amino acid substitution thereof. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 99% identity of V _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 98% identity of V _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 97% identity V _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a polypeptide that is each of the following variantsSEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 96% identity V _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 95% identity of V _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 90% identity V _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 85% identity of V _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:448 and SEQ ID NO:449 shows the sequence with at least 80% identity of V _H And V _L A zone.

In one embodiment, the anti-FAP scFv domain comprises a heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) Domain, V _H The domain comprises SEQ ID NO:450 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:451 or conservative amino acid substitutions thereof. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO: 451V having at least 99% identity to the sequence depicted _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO: 451V having at least 98% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO: 451V having at least 97% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO: 451V having at least 96% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO:451 has a sequence of at least 9V of 5% identity _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO: 451V having at least 90% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO: 451V having at least 85% identity to the sequence shown _H And V _L A zone. In one embodiment, the anti-FAP scFv domain comprises a sequence that is each of SEQ ID NO:450 and SEQ ID NO: 451V having at least 80% identity to the sequence shown _H And V _L A zone.

In one embodiment, the anti-FAP scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises a sequence selected from SEQ ID NOs: 448. SEQ ID NO:450 and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 449. SEQ ID NO:451 and sequences of conservative amino acid substitutions thereof.

In one embodiment, the anti-FAP binding domain comprises an scFv, V of OMTX-705 (available from Oncomatyryx SL) _H And/or V _L Sequences or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, which are described in U.S. patent No. 10,864,278, the disclosure of which is incorporated herein by reference in its entirety.

Exemplary FAP binding V encoding scFv Domains _H And V _L The nucleotide sequences of the domains and CDR domains are provided in table 52 and are further described in U.S. patent application publication No. US 2003/0103968 A1; US 2009/0304718 A1; US 2019/023536 A1; the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the nucleotide sequences in table 52 are codon optimized to improve protein expression.

Table 52: nucleotide sequences of exemplary FAP-binding scFv domains

In a real worldIn embodiments, the anti-FAP scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain. In one embodiment, the anti-TF scFv domain comprises V _H Domain and/or V _L Domain, V _H The domain consists of SEQ ID NO:452, a light chain variable region (V _L ) Consists of SEQ ID NO: 453. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has at least 99% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has a nucleotide encoding V having at least 98% identity to the sequence set forth in 453 _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has at least 97% identity to the nucleotide encoding V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has at least 96% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has at least 95% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has at least 90% identity to the nucleotide-encoded V _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has a nucleotide encoding V having at least 85% identity to the sequence set forth in 453 _H And/or V _L A zone. In one embodiment, the anti-TF scFv domain comprises a sequence consisting of SEQ ID NO:452 and SEQ ID NO:453 has at least 80% identity to the nucleotide-encoded V _H And/or V _L A zone. In embodiments including the preceding embodiments, SEQ ID NO:452 and/or SEQ ID NO:453 codon optimized to improve protein expression.

10. Extracellular VISTA binding domains

In one embodiment, the CCR comprises an extracellular domain, a domain capable of binding to a V domain containing T cell activating Ig inhibitor (VISTA), also known as c10orf54, PD-1H and B7-H5, which is believed, without being bound by theory, to be an immune checkpoint gene that inhibits an anti-tumor immune response. VISTA and its properties have been described in Wang et al, j.exp.med.2011,208,577-92; nowak et al, immunol. Rev.2017,276,66-79; and Deng et al, J.Immunother. Cancer,2016,4,86; the respective disclosures of which are incorporated herein by reference in their entirety. VISTA is expressed on bone marrow cells and T lymphocytes and its overexpression is associated with inhibition of early T cell activation and proliferation and reduction of cytokine production. VISTA acts as both a ligand on antigen presenting cells and a receptor on T cells. VISTA is also known to be overexpressed on certain lung cancers, such as mesothelioma and pleural mesothelioma. In one embodiment, the CCR comprises an extracellular domain that binds to human VISTA. In one embodiment, the extracellular domain binds to murine VISTA. In one embodiment, the extracellular VISTA binding domain is an scFv domain. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-VISTA V _H And V _L Domains, linkers are as described herein.

In one embodiment, the scFv domain comprises the scFv, V, of antibody 1B8, 2C12, 1a12 or 3C5 _H 、V _L Or CDR domains, their respective preparation and properties are described in U.S. patent application publication No. US 2020/0407449 A1 and incorporated herein by reference in its entirety, including V for each of 1B8, 2C12, 1A12 or 3C5 _H 、V _L And CDR domains and variants, derivatives, and fragments thereof. In one embodiment, the VISTA scFv domain of the CCR of the present invention comprises a scFv antibody comprising V described in U.S. patent application publication No. US 2020/0407449 A1 _H And V _L Domains, the disclosure of which is incorporated herein by reference in its entirety. Exemplary VISTA binding V _H And V _L The amino acid sequences of the domains are provided in table 53.

Table 53: amino acid sequence of exemplary VISTA-binding scFv domains

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In one embodiment, the anti-VISTA scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-VISTA scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:454 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:455 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has at least 99% identity of V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has at least 97% identity to V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has at least 96% identity of V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has at least 95% identity of V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has at least 90% identity to V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:454 and SEQ ID NO:455 has at least 80% identity to V _H And V _L A zone.

In one embodiment, the anti-VISTA scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody 1B1 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 456. SEQ ID NO:457 and/or SEQ ID NO:458 or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequence shown as SEQ ID NO: 459. SEQ ID NO:460 and/or SEQ ID NO:461 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown in fig.

In one embodiment, the anti-VISTA scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-VISTA scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:462 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:463 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 99% identity to the sequence shown in FIG _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 98% identity to the sequence shown in FIG. 463 _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 97% identity to the sequence shown in FIG _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 95% identity to the sequence shown in FIG. 3 _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 90% identity to the sequence shown in FIG. 463 _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:462 and SEQ ID NO:463 has a V with at least 80% identity to the sequence shown in FIG. 463 _H And V _L A zone.

In one embodiment, the anti-VISTA scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody 2C12 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 464. SEQ ID NO:465 and/or SEQ ID NO:466 or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 467. SEQ ID NO:468 and/or SEQ ID NO:469, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-VISTA scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-VISTA scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:470 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:471 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 having at least 98% identity to the sequence shown in V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 having at least 97% identity to the sequence shown in V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 having at least 96% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 has at least 95% identity of V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 has at least 90% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 has at least 85% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:470 and SEQ ID NO:471 having at least 80% identity to V _H And V _L A zone.

In one embodiment, the anti-VISTA scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody 2C12 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 472. SEQ ID NO:473 and/or SEQ ID NO:474, or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 475. SEQ ID NO:476 and/or SEQ ID NO:477, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-VISTA scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-VISTA scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:478 or a conservative amino acid substitution thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:479 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:478 and SEQ ID NO:479 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:478 and SEQ ID NO:479 has at least 98% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:478 and SEQ ID NO:479 has at least 97% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:478 and SEQ ID NO:479 has at least 96% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:478 and SEQ ID NO:479 has at least 95% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:478 and SEQ ID NO:479 has at least 90% identity V to the sequence shown _H And V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence that is each of SEQ ID NO:478 and SEQ ID NO:479 has at least 85% identity V _H And V _L A zone. In one embodiment, the anti-VISTA scFv domains comprise eachAnd (3) respectively comparing with SEQ ID NO:478 and SEQ ID NO:479 has at least 80% identity V _H And V _L A zone.

In one embodiment, the anti-VISTA scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody 3C5 or conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 480. SEQ ID NO:481 and/or SEQ ID NO:482, or conservative amino acid substitutions thereof, and/or having heavy chain CDR1, CDR2, and CDR3 domains of the sequences set forth in SEQ ID NOs: 483. SEQ ID NO:484 and/or SEQ ID NO:485 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-VISTA scFv domain comprises V _H Domain and V _L Domain, V _H The domain consists of a sequence selected from SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives, variants and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives, variants, and conservative amino acid substitutions thereof. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 99% identity V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof, V having 98% identity to the sequence _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 98% V identity to the sequence of the variant _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454.SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof, V having 97% identity to the sequence _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 97% V identity to the sequence of the variant _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof have 96% V sequence identity _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 96% V identity to the sequence of the variant _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof have 95% identity V _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 95% V identity to the sequence of the variant _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof have a sequence of 90% identity V _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 90% V identity to the sequence of the variant _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof have 85% identity V _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 85% identity V _L A zone. In one embodiment, the anti-VISTA scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 454. SEQ ID NO: 462. SEQ ID NO: 470. SEQ ID NO:478 and fragments, derivatives and variants thereof have a sequence of 80% V _H A region and a sequence selected from SEQ ID NO: 455. SEQ ID NO: 463. SEQ ID NO: 471. SEQ ID NO:479 and fragments, derivatives and variants thereof have at least 80% V identity to the sequence of the variant _L A zone.

In one embodiment, the scFv domain comprises scFv, V, of antibodies 1B8, 2C12, 1a12, 3C5, 2B7, 2C12 (H), 2C12 (L), 1C9, 1D10 _H 、V _L Or CDR domains and variants, derivatives and fragments thereof, the preparation and properties of each of which are described in U.S. patent application publication No. US 2020/0407449 A1 and are incorporated herein by reference in their entirety. Other sequences that may be employed to construct alternative VISTA binding domains suitable for use in the present invention are described in U.S. patent application publication No. US 2020/0407449 A1, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-VISTA binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent application publication No. US 2017/0306024 A1, the disclosure of which is incorporated herein by reference in its entirety.

11. Extracellular LRRC15 binding domains

In one embodiment, the CCR of the present invention comprises an extracellular domain comprising a leucine rich repeat protein 15 (LRRC 15) binding domain. LRRC15 is a cell surface protein with two known isoforms and is known to be expressed on stroma and Cancer-associated fibroblasts of many solid tumors including breast tumors, head and neck tumors, lung tumors and pancreatic tumors and directly on a subset of Cancer cells of m-yeast origin, including sarcomas, melanomas and glioblastomas, as described by Purcell et al, cancer res.2018,78,4059-72, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the CCR comprises an extracellular domain that binds to human LRRC 15. In one embodiment, the extracellular domain binds to murine LRRC 15. In one embodiment, the extracellular LRRC15 binding domain is an scFv domain. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The domain is anti-LRRC 15V _H And V _L Domains, linkers are as described herein.

In one embodiment, the anti-LRRC 15 binding domain comprises V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent No. 10,188,660, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the anti-LRRC 15 binding domain comprises V of antibody huM, huad208.4.1, huad208.12.1, huad208.14.1, hu139.10, muad210.40.9, or muad209.9.1 _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, or nucleotides encoding such sequences, each as disclosed in U.S. patent No. 10,188,660, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary LRRC15 binding scFv domains are provided in table 54.

Table 54: exemplary amino acid sequence of LRRC 15-binding scFv domains

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In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:486 or a conservation thereofAmino acid substitutions, light chain variable region (V _L ) Comprising SEQ ID NO:487 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has at least 97% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has at least 95% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has at least 90% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:486 and SEQ ID NO:487 has at least 80% identity V _H And V _L A zone.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody huM or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 488. SEQ ID NO:489 and/or SEQ ID NO:490, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 491. SEQ ID NO:492 and/or SEQ ID NO:493 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown in fig. 493.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:494 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:495 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 99% identity to the sequence set forth in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 95% identity to the sequence shown in 495 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:494 and SEQ ID NO:495 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody huad208.4.1 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 496. SEQ ID NO:497 and/or SEQ ID NO:498, and/or have conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 499. SEQ ID NO:500 and/or SEQ ID NO:501, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:502 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:503 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:502 and SEQ ID NO:503 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody huad208.12.1 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 504. SEQ ID NO:505 and/or SEQ ID NO:506 or a conservative amino acid substitution thereof, and/or a heavy chain CDR1, CDR2, and CDR3 domain having the sequence set forth in SEQ ID NO: 507. SEQ ID NO:508 and/or SEQ ID NO:509 or a conservative amino acid substitution of the light chain CDR1, CDR2, and CDR3 domains of the sequence depicted in 509.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:510 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:511 or a conservative amino acid substitution thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 99% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:510 and SEQ ID NO:511 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody huad208.14.1 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 512. SEQ ID NO:513 and/or SEQ ID NO:514, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 515. SEQ ID NO:516 and/or SEQ ID NO:517 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment The anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:518 or conservative amino acid substitutions thereof, a light chain variable region (V _L ) Comprising SEQ ID NO:519 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having at least 99% identity to V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having at least 98% identity to V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having at least 97% identity to V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having at least 96% identity V to the sequence shown in 519 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having at least 95% identity to V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having at least 90% identity to V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having a V with at least 85% identity to the sequence shown in 519 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:518 and SEQ ID NO:519 having at least 80% identity to V _H And V _L A zone.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody hu139.10 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 520. SEQ ID NO:521 and/or SEQ ID NO:522, or conservative amino acid substitutions of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 523. SEQ ID NO:524 and/or SEQ ID NO:525 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:526 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:527 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has a V with at least 99% identity to the sequence shown in 527 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has a V with at least 98% identity to the sequence shown in 527 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has a V with at least 97% identity to the sequence shown in 527 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has a V with at least 96% identity to the sequence shown in 527 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has a V with at least 95% identity to the sequence shown in 527 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has a V with at least 90% identity to the sequence shown in 527 _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has at least the sequence shown inV of 85% identity _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:526 and SEQ ID NO:527 has a V with at least 80% identity to the sequence shown in 527 _H And V _L A zone.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody muad210.40.9 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 528. SEQ ID NO:529 and/or SEQ ID NO:530 or conservative amino acid substitutions of the heavy chain CDR1, CDR2 and CDR3 domains of the sequences shown in SEQ ID NO: 531. SEQ ID NO:532 and/or SEQ ID NO:533 or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain variable region (V _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:534 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:535 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has at least 98% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has at least 97% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has a sequence as shown in seq id no V with 96% less identity _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has at least 95% identity of V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has at least 90% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has at least 85% identity V _H And V _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence that is each of SEQ ID NO:534 and SEQ ID NO:535 has at least 80% identity V _H And V _L A zone.

In one embodiment, the anti-LRRC 15 scFv domain comprises the heavy chain CDR1, CDR2, and CDR3 domains and the light chain CDR1, CDR2, and CDR3 domains of antibody muad209.9.1 or conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 536. SEQ ID NO:537 and/or SEQ ID NO:538, or conservative amino acid substitutions thereof, and/or heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 539. SEQ ID NO:540 and/or SEQ ID NO:541 or conservative amino acid substitutions of the light chain CDR1, CDR2 and CDR3 domains of the sequences shown in 541.

In one embodiment, the anti-LRRC 15 scFv domain comprises V _H Domain and V _L Domain, V _H The domain consists of a sequence selected from SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives, variants and conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising a sequence selected from the group consisting of SEQ ID NOs: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives, variants, and conservative amino acid substitutions thereof. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from SEQ ID noNO: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 99% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 99% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 98% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 98% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 97% identity to the sequence _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 97% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 96% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 96% identity to the sequence of the fragment, derivative or variant _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ IDNO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 95% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 95% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 90% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 90% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 85% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 85% identity to the sequence of the fragment _L A zone. In one embodiment, the anti-LRRC 15 scFv domain comprises a sequence selected from the group consisting of SEQ ID NOs: 486. SEQ ID NO: 494. SEQ ID NO: 502. SEQ ID NO: 510. SEQ ID NO: 518. SEQ ID NO: 526. SEQ ID NO:534 and fragments, derivatives and variants thereof, and V having 80% identity to the sequence of the variant _H A region and a sequence selected from SEQ ID NO: 487. SEQ ID NO: 495. SEQ ID NO: 503. SEQ ID NO: 511. SEQ ID NO: 519. SEQ ID NO: 527. SEQ ID NO:535 and fragments, derivatives and variants thereof, and V having at least 80% identity to the sequence of the fragment _L A zone.

12. Extracellular B7-H3 binding domains

In one embodiment, a CCR of the invention comprises an extracellular domain comprising a B7-H3 (also known as CD 276) binding structureDomain. B7-H3 (B7 homology 3) is a cell surface glycoprotein expressed on antigen presenting cells and is known to play a role in both immune evasion and cancer progression, as described by Castellanos et al, am.j.clin.exp.immunol.2017,6,66-75, the disclosure of which is incorporated herein by reference in its entirety. Exon repeats in humans lead to the expression of two B7-H3 isoforms having a single IgV-IgC-like domain (2 IgB7-H3 isoform) or IgV-IgC-like domain (4 IgB7-H3 isoform) and containing several conserved cysteine residues. The major B7-H3 isomer in human tissues and cell lines is the 4IgB7-H3 isomer, as described by Steinberger et al, J.Immunol.2004,172,2352-59, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the CCR comprises an extracellular domain that binds to human B7-H3. In one embodiment, the CCR comprises an extracellular domain that binds to human 4IgB 7-H3. In one embodiment, the CCR comprises an extracellular domain that binds to human 2IgB 7-H3. In one embodiment, the extracellular domain binds to murine B7-H3. In one embodiment, the extracellular B7-H3 binding domain is a scFv domain. In one embodiment, the CCR of the invention comprises a construct as shown in FIG. 34, V _H And V _L The structural domain is anti-B7-H3V _H And V _L Domains, linkers are as described herein.

In one embodiment, the anti-B7-H3 binding domain comprises V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent No. 10,730,945, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the anti-B7-H3 binding domain comprises a humanized or murine (including variants thereof) antibody BRCA84D (including BRCA84D-1 and BRCA84D-2 and variants thereof), BRCA68D, BRCA69D, PRCA157, TES7, OVCA22, GB8 or SG 27V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, or nucleotides encoding such sequences, each as disclosed in U.S. patent No. 10,730,945, the disclosure of which is incorporated herein by reference in its entirety. The amino acid sequences of exemplary B7-H3 binding scFv domains are provided in Table 55.

Table 55: amino acid sequence of exemplary B7-H3 binding scFv Domain

In one embodiment, the anti-B7-H3 scFv domain comprises the heavy chain variable region (V) of the antibodies BRCA84D (including BRCA84D-1 and BRCA84D-2 and variants thereof), BRCA68D, BRCA69D, PRCA157, TES7, OVCA22, GB8 or SG27 (including humanized or murine variants) _H ) Domain and/or light chain variable region (V _L ) A domain or variant, derivative, fragment or conservative amino acid substitution thereof. In one embodiment, the anti-B7-H3 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:542 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:543 or conservative amino acid substitutions thereof. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 99% identity to the sequence shown in figure _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 98% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 97% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 95% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 90% identity to the sequence shown in figure _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises each of the followingGraph-cut with SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 85% identity to the sequence shown in figure _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:542 and SEQ ID NO:543 has a V with at least 80% identity to the sequence shown in seq id no _H And V _L A zone.

In one embodiment, the anti-B7-H3 scFv domain comprises the heavy chain CDR1, CDR2 and CDR3 domains and the light chain CDR1, CDR2 and CDR3 domains of antibodies BRCA84D (including BRCA84D-1 and BRCA84D-2 and variants thereof), BRCA68D, BRCA69D, PRCA157, TES7, OVCA22, GB8 or SG27 (including both humanized and murine variants), or conservative amino acid substitutions thereof. In one embodiment, the anti-B7-H3 scFv domain comprises a polypeptide having the sequence set forth in SEQ ID NO: 544. SEQ ID NO:545 and/or SEQ ID NO:546, or a conservative amino acid substitution of the heavy chain CDR1, CDR2, and CDR3 domains of the sequences shown in SEQ ID NO: 547. SEQ ID NO:548 and/or SEQ ID NO:549, or conservative amino acid substitutions of the light chain CDR1, CDR2, and CDR3 domains of the sequences shown.

In one embodiment, the anti-B7-H3 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises SEQ ID NO:550 or conservative amino acid substitutions thereof, light chain variable region (V _L ) Comprising SEQ ID NO:551 or a conservative amino acid substitution thereof. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has at least 99% identity V _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has a V with at least 98% identity to the sequence shown in 551 _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has at least 97% identity V _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has a V with at least 96% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has at least 95% identity V to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has a V with at least 90% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has a V with at least 85% identity to the sequence shown in seq id no _H And V _L A zone. In one embodiment, the anti-B7-H3 scFv domain comprises a sequence that hybridizes to SEQ ID NO:550 and SEQ ID NO:551 has at least 80% identity V _H And V _L A zone.

In one embodiment, the anti-B7-H3 scFv domain comprises V _H Domain and V _L Domain, V _H The domain comprises a sequence selected from BRCA84D, BRCA69D, PRCA157, BRCA84D-1, hBRCA84D-2VH, hBRCA84D-3VH, hBRCA84D-4VH, chBRCA84D, or a conservative amino acid substitution thereof, a light chain variable region (V _L ) Comprising a sequence selected from BRCA84D, BRCA69D, PRCA157, BRCA84D-1, hBRCA84D-2VL, hBRCA84D-3VL, hBRCA84D-4VL, hBRCA84D-5VL, hBRCA84D-6VL, chBRCA84D, or a conservative amino acid substitution thereof, each as disclosed in U.S. Pat. No. 10,730,945, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-B7-H3 binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent No. 9,371,395, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-B7-H3 binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent No. 10,501,544, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the anti-B7-H3 binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, as disclosed in U.S. patent application publication No. US 2020/0338209 A1, the disclosure of which is incorporated herein by reference in its entirety.

13. Other extracellular binding domains

In one embodiment, a CCR of the present invention comprises an extracellular domain comprising a CD44 binding domain. In one embodiment, the anti-CD 44 binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, such as U.S. patent No. 7,361,347; 9,388,249 and 11,220,544 and U.S. patent application publication No. US 2004/0048319 A1; US 2005/0214283 A1; US 2007/0237761 A1; US 2010/0092484 A1; US 2012/0201751 A1 and US 2020/0291113 A1, the respective disclosures of which are incorporated herein by reference in their entirety. Other suitable anti-CD 44 binding domains known in the art may also be used.

In one embodiment, a CCR of the invention comprises an extracellular domain comprising a CD40 binding domain. In one embodiment, the anti-CD 40 binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, such as international patent application publication No. WO 2018/027025 A1; U.S. patent application publication No. US 2021/0188992 A1; and 2015/01108783 A1; and U.S. patent nos. 11,001,637 and 10,577,425, the disclosures of each of which are incorporated herein by reference in their entirety. The CD40 binding domain also includes the binding or extracellular portion of the CD40 ligand (CD 40L) domain. Other suitable anti-CD 40 binding domains known in the art may also be used.

In one embodiment, the CCR of the present invention comprises an extracellular domain comprising an ALCAM (CD 166) binding domain. In one embodiment, the anti-ALCAM (anti-CD 166) binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, e.g.U.S. Pat. No. 9,388,249And 11,220,544 and U.S. patent application publication Nos. US 2004/0048319 A1 and US 2020/0291113 A1, the respective disclosures of which are incorporated herein by reference in their entirety. Other suitable anti-ALCAM binding domains known in the art may also be used.

In one embodiment, a CCR of the invention comprises an extracellular domain comprising an IL-13Rα binding domain. In one embodiment, a CCR of the invention comprises an extracellular domain comprising an IL-13Rα1 binding domain. In one embodiment, a CCR of the invention comprises an extracellular domain comprising an IL-13Rα2 binding domain. In one embodiment, the anti-IL-13 Rα binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, such as U.S. patent No. 6,428,788; 7,994,302; 8,221,755; 9,315,575; 8,318,910; 9,650,438; 9,828,428; 9,587,026; and U.S. patent application publication No. US 2019/0008966 A1; US 2021/0000875 A1; and US 2019/0359723 A1, the respective disclosures of which are incorporated herein by reference in their entirety. Other suitable anti-IL-13 Rα, IL-13Rα1 or IL-13Rα2 binding domains known in the art may also be used.

In one embodiment, a CCR of the present invention comprises an extracellular domain comprising a transforming growth factor beta receptor (tgfβr) binding domain. In one embodiment, a CCR of the present invention comprises an extracellular domain comprising a tgfbetarii (also referred to herein as tgfbetar 2) binding domain. In one embodiment, the anti-TGF-beta R binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2, and/or CDR3 sequences, such as U.S. patent nos. 6,201,108 and 7,579,186; U.S. patent application publication No. US 2012/0177666 A1; and International patent application publication No. WO 2021/133167 A1, the respective disclosures of which are incorporated herein by reference in their entirety. TGF-beta R binding domains also include binding or extracellular portions of TGF-beta domains. May also use the artOther suitable anti-tgfβr binding domains known in the art.

In one embodiment, a CCR of the present invention comprises an extracellular domain comprising a transforming growth factor beta (tgfβ) binding domain. In one embodiment, the anti-TGF-beta binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, such as U.S. patent No. 10,947,303; 9,714,285; and 9,676,863 and U.S. patent application publication No. US 2021/0061897 A1, the respective disclosures of which are incorporated herein by reference in their entirety. TGF-beta binding-domains also include the binding or extracellular portion of TGF-beta R domains (including TGF-beta RII domains). Other suitable anti-tgfβ binding domains known in the art may also be used.

In one embodiment, the CCR of the present invention comprises an extracellular domain comprising a FAS (or FAS) binding domain. In one embodiment, the anti-FAS binding domain comprises scFv, V _H And/or V _L Sequences, or heavy and/or light chain CDR1, CDR2 and/or CDR3 sequences, such as U.S. patent No. 6,086,877; 6,746,673; and 6,972,323 and U.S. patent application publication Nos. US 2006/0083738 A1 and US 2010/023157 A1, the respective disclosures of which are incorporated herein by reference in their entirety. Other suitable anti-FAS binding domains known in the art may also be used.

In one embodiment, any of the extracellular domains disclosed herein can be used to produce a bi-epitope binding CCR construct as described elsewhere herein.

B. Transmembrane and hinge domains

In one embodiment, the CCR comprises a transmembrane domain. In one embodiment, one end of the transmembrane domain is linked to the extracellular domain of CCR and the other end is linked to at least one intracellular domain of CCR. In one embodiment, one end of the transmembrane domain is linked to the extracellular domain of CCR and the other end is linked to at least one intracellular domain of CCR. In another embodiment, the CCR is a transmembrane domain designed to comprise a spacer or hinge domain fused to the CCR, which spacer or hinge domain itself is fused to the extracellular domain of the CCR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CCR. In some embodiments, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of the domain to the transmembrane domain of the same or a different surface membrane protein, e.g., to minimize interactions with other members of the receptor complex, without being bound by any theory.

The transmembrane domains used in CCR of the present invention may be derived from or comprise at least the transmembrane region of the α, β or ζ chain (including cd3ζ), cd3ε, CD4, CD5, CD8 (including cd8α), CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2rα, IL-2rβ and IL-2rγ of the T cell receptor. In some embodiments, the transmembrane domain may be synthetic and predominantly comprise hydrophobic residues such as leucine and valine. In some embodiments, the three amino acids phenylalanine, tryptophan, and valine will be located at each end of the synthetic transmembrane domain. Alternatively, in some embodiments, a short oligopeptide or polypeptide linker (between 2 and 10 amino acids in length) may form a linkage between the transmembrane domain and the intracellular domain of CCR, in some embodiments, this linker may comprise glycine-serine double amino acids as described herein. Suitable, non-limiting transmembrane domains for CCR constructs useful in the present invention are shown in table 56.

Table 56: amino acid sequences of exemplary transmembrane and hinge domains

In one embodiment, the CCR of the present invention comprises a transmembrane domain and optionally a hinge domain. In one embodiment, the CCR of the present invention comprises a transmembrane domain and an optional hinge domain, as shown in fig. 34. In one embodiment, the CCR of the invention comprises a transmembrane domain selected from the transmembrane region of the alpha, beta or zeta chain of a T cell receptor (including CD3 zeta), CD3 epsilon, CD4, CD5, CD8 (including CD8 alpha), CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2 Ralpha, IL-2 Rbeta, IL-2 Rgamma and variants, fragments and derivatives thereof. In some embodiments, the transmembrane domain of a CCR of the invention may be recombinant, in which case it will predominantly comprise hydrophobic residues such as leucine and valine, in some embodiments the three amino acids phenylalanine, tryptophan and valine may be located at each end of the recombinant transmembrane domain. In one embodiment, the CCR comprises a hinge domain. In one embodiment, the hinge domain is a spacer domain. In one embodiment, the CCR of the present invention comprises a hinge domain or spacer domain derived from a human protein. In one embodiment, the hinge domain or spacer domain is located between and linked to the extracellular domain and the transmembrane domain. In some embodiments, a CCR of the invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain, but does not comprise a hinge domain. In some embodiments, a CCR of the invention comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular domain. In some embodiments, a CCR of the invention comprises an extracellular domain and an intracellular domain comprising a transmembrane domain, with or without a hinge domain.

In certain instances, the transmembrane domain may be attached to the extracellular region of CCR by a hinge domain (e.g., a hinge region from a human protein). For example, in some embodiments, CCR of the present invention include human Ig (immunoglobulin) hinges, such as IgG4 hinges or CD8 a hinges. In one embodiment, the CCR of the invention comprises a hinge domain selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2 Ralpha, IL-2 Rbeta, IL-2 Rgamma, and variants, fragments, and derivatives thereof.

In one embodiment, a CCR of the invention comprises a PD-1 transmembrane domain comprising the amino acid sequence of SEQ ID NO:552 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:552, has at least 99% identity to the sequence set forth in SEQ ID NO:552, has at least 98% identity to the sequence given in SEQ ID NO:552, has at least 97% identity to the sequence given in SEQ ID NO:552, has at least 96% identity to the sequence given in SEQ ID NO:552, has at least 95% identity to the sequence given in SEQ ID NO:552, has at least 90% identity to the sequence given in SEQ ID NO:552, or a sequence having at least 85% identity to SEQ ID NO:552 has an amino acid sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:553, or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof, or an amino acid sequence that hybridizes with SEQ ID NO:553, has at least 99% identity to the sequence given in SEQ ID NO:553, has at least 98% identity to the sequence given in SEQ ID NO:553, has at least 97% identity to the sequence given in SEQ ID NO:553, has at least 96% identity to the sequence given in SEQ ID NO:553, has at least 95% identity to the sequence given in SEQ ID NO:553, has at least 90% identity to the sequence given in SEQ ID NO:553, or a sequence having at least 85% identity to SEQ ID NO:553 has an amino acid sequence with at least 80% identity.

In one embodiment, a CCR of the invention comprises a CD27 transmembrane domain comprising the amino acid sequence of SEQ ID NO:554 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes with SEQ ID NO:554, has at least 99% identity to the sequence set forth in SEQ ID NO:554, has at least 98% identity to the sequence set forth in SEQ ID NO:554, has at least 97% identity to the sequence set forth in SEQ ID NO:554, has at least 96% identity to the sequence set forth in SEQ ID NO:554, has at least 95% identity to the sequence set forth in SEQ ID NO:554, has at least 90% identity to the sequence set forth in SEQ ID NO:554, or a sequence having at least 85% identity to SEQ ID NO:554, has an amino acid sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises a CD8 a transmembrane domain comprising the amino acid sequence of SEQ ID NO:555 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes with SEQ ID NO:555, having at least 99% identity to the sequence set forth in SEQ ID NO:555, having at least 98% identity to the sequence set forth in SEQ ID NO:555, having at least 97% identity to the sequence set forth in SEQ ID NO:555, having at least 96% identity to the sequence set forth in SEQ ID NO:555, having at least 95% identity to the sequence set forth in SEQ ID NO:555, having at least 90% identity to the sequence set forth in SEQ ID NO:555, or a sequence having at least 85% identity to SEQ ID NO:555, the amino acid sequence having at least 80% identity.

In one embodiment, the CCR of the present invention comprises a CD8 a hinge domain comprising the amino acid sequence of SEQ ID NO:556 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:556, has at least 99% identity to the sequence given in SEQ ID NO:556, has at least 98% identity to the sequence given in SEQ ID NO:556, has at least 97% identity to the sequence given in SEQ ID NO:556, has at least 96% identity to the sequence given in SEQ ID NO:556, has at least 95% identity to the sequence given in SEQ ID NO:556, has at least 90% identity to the sequence given in SEQ ID NO:556, or a sequence having at least 85% identity to SEQ ID NO:556 has an amino acid sequence with at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-2rβ transmembrane domain comprising the amino acid sequence of SEQ ID NO:557 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:557, has at least 99% identity to the sequence set forth in SEQ ID NO:557, has at least 98% identity to the sequence set forth in SEQ ID NO:557, has at least 97% identity to the sequence set forth in SEQ ID NO:557, has at least 96% identity to the sequence set forth in SEQ ID NO:557, has at least 95% identity to the sequence set forth in SEQ ID NO:557, has at least 90% identity to the sequence set forth in SEQ ID NO:557, or a sequence having at least 85% identity to SEQ ID NO:557 has an amino acid sequence with at least 80% identity.

In one embodiment, the CCR of the present invention comprises an IgG1 transmembrane and hinge domain comprising the amino acid sequence of SEQ ID NO:558 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes with SEQ ID NO:558, has at least 99% identity to the sequence set forth in SEQ ID NO:558, has at least 98% identity to the sequence set forth in SEQ ID NO:558, has at least 97% identity to SEQ ID NO:558, has at least 96% identity to the sequence set forth in SEQ ID NO:558, has at least 95% identity to the sequence set forth in SEQ ID NO:558, has at least 90% identity to the sequence set forth in SEQ ID NO:558, or a sequence having at least 85% identity to SEQ ID NO:558 has an amino acid sequence having at least 80% identity.

In one embodiment, the CCR of the present invention comprises an IgG1 hinge domain comprising the amino acid sequence of SEQ ID NO:559 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:559, has at least 99% identity to the sequence set forth in SEQ ID NO:559, has at least 98% identity to the sequence set forth in SEQ ID NO:559, has at least 97% identity to the sequence set forth in SEQ ID NO:559, has at least 96% identity to the sequence set forth in SEQ ID NO:559, has at least 95% identity to the sequence set forth in SEQ ID NO:559, has at least 90% identity to the sequence set forth in SEQ ID NO:559, or a sequence having at least 85% identity to SEQ ID NO:559 has an amino acid sequence with at least 80% identity.

In one embodiment, the CCR of the present invention comprises an IgG4 hinge domain comprising the amino acid sequence of SEQ ID NO:560 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:560 has at least 99% identity to the sequence set forth in SEQ ID NO:560 has at least 98% identity to the sequence set forth in SEQ ID NO:560 has at least 97% identity to the sequence set forth in SEQ ID NO:560 has at least 96% identity to the sequence set forth in SEQ ID NO:560 has at least 95% identity to the sequence set forth in SEQ ID NO:560 has at least 90% identity to the sequence set forth in SEQ ID NO:560, or a sequence having at least 85% identity to SEQ ID NO:560 has an amino acid sequence having at least 80% identity.

In one embodiment, the CCR of the present invention comprises an IgD hinge domain comprising the amino acid sequence of SEQ ID NO:561 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:561 has at least 99% identity to the sequence set forth in SEQ ID NO:561 has at least 98% identity to the sequence set forth in SEQ ID NO:561 has at least 97% identity to the sequence set forth in SEQ ID NO:561 has at least 96% identity to the sequence set forth in SEQ ID NO:561 has at least 95% identity to the sequence set forth in SEQ ID NO:561 has at least 90% identity to the sequence set forth in SEQ ID NO:561, or a sequence having at least 85% identity to SEQ ID NO:561 has an amino acid sequence with at least 80% identity.

Nucleotide sequences encoding exemplary transmembrane and hinge domains for CCR's of the present invention are provided in table 57. In one embodiment, the nucleotide sequence of table 57 is codon optimized to improve protein expression.

Table 57: nucleotide sequences of exemplary transmembrane and hinge domains

In one embodiment, the transmembrane and/or hinge domain comprises a domain encoded by a nucleotide sequence selected from the group consisting of the α, β or ζ chain, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2rα, IL-2rβ, IL-2rγ, and variants, fragments, and derivatives thereof of the T cell receptor. In one embodiment, the transmembrane and/or hinge domain consists of SEQ ID NO:562, and the sequence code shown in 562. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 have a nucleotide coding of at least 99% identity. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 have a nucleotide code of at least 98% identity. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 has a nucleotide encoding at least 97% identical. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 have a nucleotide coding of at least 96% identity. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 have a nucleotide encoding at least 95% identical. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 have a nucleotide code with at least 90% identity. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 has a nucleotide encoding at least 85% identical. In one embodiment, the transmembrane and/or hinge domain consists of a sequence selected from SEQ ID NOs: 562. SEQ ID NO: 563. SEQ ID NO: 564. SEQ ID NO: 565. SEQ ID NO: 566. SEQ ID NO: 567. SEQ ID NO: 568. SEQ ID NO: 569. SEQ ID NO:570 and SEQ ID NO:571 have a nucleotide coding of at least 80% identity.

In some embodiments, the CCR of the present invention comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular domain, the hinge domain being linked to the transmembrane domain by a linker comprising two to forty amino acids. In some embodiments, a CCR of the invention comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular domain, the hinge domain being encoded by a sequence selected from the group consisting of SEQ ID NOs: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72. SEQ ID NO: 74. SEQ ID NO: 75. SEQ ID NO: 76. SEQ ID NO: 238. SEQ ID NO: 239. SEQ ID NO: 240. SEQ ID NO: 241. SEQ ID NO: 242. SEQ ID NO: 243. SEQ ID NO:587 and fragments, variants and derivatives thereof are linked to the transmembrane domain. Alternative linkers as disclosed herein or known in the art, such as those described in U.S. patent No. 9,394,368, the disclosure of which is incorporated herein by reference in its entirety, may also be used.

In some embodiments, a CCR of the invention comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising an scFv domain, and the hinge domain is linked to the scFv domain by a linker comprising two to forty amino acids. In some embodiments, a CCR of the invention comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising an scFv domain, and the hinge domain is encoded by a sequence selected from the group consisting of SEQ ID NOs: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72. SEQ ID NO: 74. SEQ ID NO: 75. SEQ ID NO: 76. SEQ ID NO: 238. SEQ ID NO: 239. SEQ ID NO: 240. SEQ ID NO: 241. SEQ ID NO: 242. SEQ ID NO: 243. SEQ ID NO:587 and fragments, variants and derivatives thereof are linked to scFv domains. Alternative linkers as disclosed herein or known in the art, such as those described in U.S. patent No. 9,394,368, the disclosure of which is incorporated herein by reference in its entirety, may also be used.

In some embodiments, a CCR of the invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising an scFv domain, and the transmembrane domain is linked to the scFv domain by a linker comprising two to forty amino acids. In some embodiments, a CCR of the invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising an scFv domain, and the transmembrane domain is encoded by a sequence selected from the group consisting of SEQ ID NOs: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72. SEQ ID NO: 74. SEQ ID NO: 75. SEQ ID NO: 76. SEQ ID NO: 238. SEQ ID NO: 239. SEQ ID NO: 240. SEQ ID NO: 241. SEQ ID NO: 242. SEQ ID NO: 243. SEQ ID NO:587 and fragments, variants and derivatives thereof are linked to scFv domains. Alternative linkers as disclosed herein or known in the art, such as those described in U.S. patent No. 9,394,368, the disclosure of which is incorporated herein by reference in its entirety, may also be used.

In one embodiment, the CCR of the present invention comprises a transmembrane domain comprising a CD40L (CD 154) transmembrane domain. Suitable CD40L transmembrane domains are described in Aloui et al, int.J.mol.Sci.2014,15 (12), 22342-22364, and U.S. Pat. No. 10,287,354, the disclosures of each of which are incorporated herein by reference in their entirety.

C. Intracellular domains

In one embodiment, the CCR comprises an intracellular domain, also referred to herein as a signaling domain, co-stimulatory domain, or intracellular domain (endodomain). The intracellular domains may provide costimulatory activation signals to T cells useful in the invention or may activate alternative signaling pathways in T cells. In one embodiment, the CCR comprises a cytoplasmic intracellular domain. In one embodiment, the CCR comprises cytoplasmic and transmembrane intracellular domains. In some embodiments, the intracellular domain is selected from the group consisting of the α, β, or ζ chain of a T cell receptor (including CD3 ζ), CD3 ε, CD4, CD5, CD8 (including CD8 α), CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134 (OX 40), CD137 (TNFRSF 9,4-1 BB), CD150 (SLAM), CD270 (HVEM), CD278 (ICOS), CD357 (GITR), ephB6, STAT3, IL-2R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL18-R1, IL-18RAP, IL-21R, and LTBR (lymphotoxin β receptor, TNFRSF 3). In some embodiments, the intracellular domain employs the full length of one of the foregoing protein sequences (excluding the signal peptide). In some embodiments, the intracellular domain employs a truncated portion of one of the foregoing protein sequences. In one embodiment, the intracellular domain is a full length CD28 sequence. Suitable, non-limiting intracellular domains for use in CCR constructs of the invention are shown in table 58.

Table 58: amino acid sequence of exemplary intracellular domains

In one embodiment, a CCR of the invention comprises a CD28 intracellular domain comprising the amino acid sequence of SEQ ID NO:572 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:572 has at least 99% identity to the sequence set forth in SEQ ID NO:572 has at least 98% identity to the sequence set forth in SEQ ID NO:572 has at least 97% identity to the sequence set forth in SEQ ID NO:572 has at least 96% identity to the sequence set forth in SEQ ID NO:572 has at least 95% identity to the sequence set forth in SEQ ID NO:572 has at least 90% identity to the sequence set forth in SEQ ID NO:572, or a sequence having at least 85% identity to SEQ ID NO:572 to a sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises a CD134 (OX 40) intracellular domain comprising the amino acid sequence of SEQ ID NO:573 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:573, has at least 99% identity to the sequence set forth in SEQ ID NO:573, has at least 98% identity to the sequence set forth in SEQ ID NO:573, has at least 97% identity to the sequence set forth in SEQ ID NO:573, has at least 96% identity to the sequence set forth in SEQ ID NO:573, has at least 95% identity to the sequence set forth in SEQ ID NO:573, has at least 90% identity to the sequence set forth in SEQ ID NO:573, or a sequence having at least 85% identity to SEQ ID NO:573 has an amino acid sequence that is at least 80% identical.

In one embodiment, a CCR of the invention comprises a CD278 (ICOS) intracellular domain comprising the amino acid sequence of SEQ ID NO:574 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:574, has at least 99% identity to the sequence set forth in SEQ ID NO:574, has at least 98% identity to a sequence set forth in SEQ ID NO:574, has at least 97% identity to the sequence given in SEQ ID NO:574, has at least 96% identity to a sequence set forth in SEQ ID NO:574, has at least 95% identity to the sequence set forth in SEQ ID NO:574, has at least 90% identity to the sequence set forth in SEQ ID NO:574, or a sequence having at least 85% identity to SEQ ID NO:574 has an amino acid sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises a CD137 (4-1 BB) intracellular domain comprising the amino acid sequence of SEQ ID NO:575 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof, or an amino acid sequence that hybridizes to SEQ ID NO:575, has at least 99% identity to the sequence set forth in SEQ ID NO:575, has at least 98% identity to the sequence set forth in SEQ ID NO:575, has at least 97% identity to the sequence set forth in SEQ ID NO:575, has at least 96% identity to the sequence set forth in SEQ ID NO:575, has at least 95% identity to the sequence set forth in SEQ ID NO:575, has at least 90% identity to the sequence set forth in SEQ ID NO:575, or a sequence having at least 85% identity to SEQ ID NO:575, the sequence given by seq id No. s has an amino acid sequence of at least 80% identity.

In one embodiment, a CCR of the invention comprises a CD27 intracellular domain comprising the amino acid sequence of SEQ ID NO:576 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes with SEQ ID NO:576, having at least 99% identity to the sequence set forth in SEQ ID NO:576, having at least 98% identity to the sequence set forth in SEQ ID NO:576, having at least 97% identity to the sequence set forth in SEQ ID NO:576, having at least 96% identity to the sequence set forth in SEQ ID NO:576, having at least 95% identity to the sequence set forth in SEQ ID NO:576, having at least 90% identity to the sequence set forth in SEQ ID NO:576, or a sequence having at least 85% identity to SEQ ID NO:576, an amino acid sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises a cd3ζ intracellular domain comprising an amino acid sequence of SEQ ID NO:577 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:577, has at least 99% identity to the sequence set forth in SEQ ID NO:577 has at least 98% identity to the sequence set forth in SEQ ID NO:577, has at least 97% identity to the sequence set forth in SEQ ID NO:577, has at least 96% identity to the sequence set forth in SEQ ID NO:577, has at least 95% identity to the sequence set forth in SEQ ID NO:577, has at least 90% identity to the sequence set forth in SEQ ID NO:577, or a sequence having at least 85% identity to SEQ ID NO:577 has an amino acid sequence that is at least 80% identical.

In one embodiment, a CCR of the invention comprises an IL-2rβ intracellular domain comprising the amino acid sequence of SEQ ID NO:578 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:578, has at least 99% identity to the sequence given in SEQ ID NO:578, has at least 98% identity to the sequence given in SEQ ID NO:578, has at least 97% identity to the sequence given in SEQ ID NO:578, has at least 96% identity to the sequence given in SEQ ID NO:578, has at least 95% identity to the sequence given in SEQ ID NO:578, has at least 90% identity to the sequence given in SEQ ID NO:578, or a sequence having at least 85% identity to SEQ ID NO:578, the sequence given has an amino acid sequence with at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-2rγ intracellular domain comprising the amino acid sequence of SEQ ID NO:579 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:579, has at least 99% identity to the sequence set forth in SEQ ID NO:579 has at least 98% identity to the sequence set forth in SEQ ID NO:579, has at least 97% identity to the sequence set forth in SEQ ID NO:579 has at least 96% identity to the sequence set forth in SEQ ID NO:579, has at least 95% identity to the sequence set forth in SEQ ID NO:579, has at least 90% identity to the sequence set forth in SEQ ID NO:579, or a sequence having at least 85% identity to SEQ ID NO:579 has an amino acid sequence that is at least 80% identical.

In one embodiment, a CCR of the invention comprises an IL-18R1 intracellular domain comprising the amino acid sequence of SEQ ID NO:580 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:580, has at least 99% identity to the sequence set forth in SEQ ID NO:580, has at least 98% identity to the sequence set forth in SEQ ID NO:580, has at least 97% identity to the sequence set forth in SEQ ID NO:580, has at least 96% identity to the sequence set forth in SEQ ID NO:580, has at least 95% identity to the sequence set forth in SEQ ID NO:580, has at least 90% identity to the sequence set forth in SEQ ID NO:580, or a sequence having at least 85% identity to SEQ ID NO:580 has an amino acid sequence with at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-7rα intracellular domain comprising the amino acid sequence of SEQ ID NO:581 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:581, having at least 99% identity to the sequence set forth in SEQ ID NO:581, having at least 98% identity to the sequence set forth in SEQ ID NO:581, having at least 97% identity to the sequence set forth in SEQ ID NO:581, having at least 96% identity to the sequence set forth in SEQ ID NO:581, having at least 95% identity to the sequence set forth in SEQ ID NO:581, having at least 90% identity to the sequence set forth in SEQ ID NO:581, or a sequence having at least 85% identity to SEQ ID NO:581 to seq id no.

In one embodiment, a CCR of the invention comprises an IL-12R1 intracellular domain comprising the amino acid sequence of SEQ ID NO:582 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:582, having at least 99% identity to SEQ ID NO:582, having at least 98% identity to SEQ ID NO:582, having at least 97% identity to SEQ ID NO:582, having at least 96% identity to SEQ ID NO:582, having at least 95% identity to SEQ ID NO:582, having at least 90% identity to SEQ ID NO:582, or a sequence having at least 85% identity to SEQ ID NO:582 has an amino acid sequence with at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-12R2 intracellular domain comprising the amino acid sequence of SEQ ID NO:583 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:583 has at least 99% identity to the sequence set forth in SEQ ID NO:583 has at least 98% identity to the sequence set forth in SEQ ID NO:583 has at least 97% identity to the sequence set forth in SEQ ID NO:583 has at least 96% identity to the sequence set forth in SEQ ID NO:583 has at least 95% identity to the sequence set forth in SEQ ID NO:583 has at least 90% identity to the sequence set forth in SEQ ID NO:583, or a sequence having at least 85% identity to SEQ ID NO:583 has an amino acid sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-15 ra intracellular domain comprising the amino acid sequence of SEQ ID NO:584 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes to SEQ ID NO:584, has at least 99% identity to the sequence set forth in SEQ ID NO:584, has at least 98% identity to the sequence set forth in SEQ ID NO:584, has at least 97% identity to the sequence set forth in SEQ ID NO:584, has at least 96% identity to the sequence set forth in SEQ ID NO:584, has at least 95% identity to the sequence set forth in SEQ ID NO:584, has at least 90% identity to the sequence set forth in SEQ ID NO:584, or a sequence having at least 85% identity to SEQ ID NO:584 has an amino acid sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-21R intracellular domain comprising the amino acid sequence of SEQ ID NO:585 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:585, has at least 99% identity to the sequence set forth in SEQ ID NO:585 has at least 98% identity to the sequence set forth in SEQ ID NO:585, having at least 97% identity to the sequence set forth in SEQ ID NO:585, having at least 96% identity to the sequence set forth in SEQ ID NO:585, has at least 95% identity to the sequence set forth in SEQ ID NO:585, has at least 90% identity to the sequence set forth in SEQ ID NO:585, or a sequence having at least 85% identity to SEQ ID NO:585, an amino acid sequence having at least 80% identity to the sequence set forth.

In one embodiment, the CCR of the present invention comprises an LTBR intracellular domain comprising the amino acid sequence of SEQ ID NO:586 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:586 has at least 99% identity to the sequence set forth in SEQ ID NO:586 has at least 98% identity to the sequence set forth in SEQ ID NO:586 has at least 97% identity to the sequence set forth in SEQ ID NO:586, has at least 96% identity to the sequence set forth in SEQ ID NO:586 has at least 95% identity to the sequence set forth in SEQ ID NO:586, has at least 90% identity to the sequence set forth in SEQ ID NO:586, or a sequence having at least 85% identity to SEQ ID NO:586 has an amino acid sequence with at least 80% identity.

In some embodiments, the intracellular domain may be directly linked to the transmembrane domain. In some embodiments, the intracellular domain can be linked to the transmembrane domain by a linker. Alternatively, in some embodiments, a short oligopeptide or polypeptide linker (between 2 and 10 amino acids in length) may form a linkage between the transmembrane domain and the intracellular domain of CCR, in some embodiments, this linker may comprise a glycine-serine double amino acid or other alternative linker as described herein. In some embodiments, the linker comprises a sequence selected from SEQ ID NOs: 587. SEQ ID NO: 238. SEQ ID NO: 239. SEQ ID NO: 240. SEQ ID NO: 241. SEQ ID NO: 242. SEQ ID NO: 243. SEQ ID NO: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72. SEQ ID NO: 74. SEQ ID NO:75 and SEQ ID NO: 76.

Nucleotide sequences encoding exemplary intracellular domains for CCR's of the present invention are provided in table 59. In one embodiment, the nucleotide sequences in table 59 are codon optimized to improve protein expression.

Table 59: nucleotide sequences of exemplary intracellular signaling domains

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In one embodiment, a CCR of the invention comprises a CD28 intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:588, or a sequence identical to SEQ ID NO:588, has at least 99% identity to the sequence set forth in SEQ ID NO:588, has at least 98% identity to a sequence set forth in SEQ ID NO:588, has at least 97% identity to the sequence set forth in SEQ ID NO:588, has at least 96% identity to the sequence set forth in SEQ ID NO:588, has at least 95% identity to the sequence set forth in SEQ ID NO:588, has at least 90% identity to the sequence set forth in SEQ ID NO:588 or a sequence which has at least 85% identity to SEQ ID NO:588, a sequence having at least 80% identity to the sequence.

In one embodiment, the CCR of the present invention comprises a CD134 intracellular domain, also known as OX40 intracellular domain, consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:589, or a sequence identical to SEQ ID NO:589 has at least 99% identity to the sequence set forth in SEQ ID NO:589 has at least 98% identity to the sequence set forth in SEQ ID NO:589 has at least 97% identity to the sequence set forth in SEQ ID NO:589 has at least 96% identity to the sequence set forth in SEQ ID NO:589 has at least 95% identity to the sequence set forth in SEQ ID NO:589 has at least 90% identity to the sequence set forth in SEQ ID NO:589 has at least 85% identity or is identical to the sequence set forth in SEQ ID NO:589, a sequence having at least 80% identity to the sequence.

In one embodiment, the CCR of the present invention comprises a CD278 intracellular domain, also known as ICOS intracellular domain, consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:590, or a sequence identical to SEQ ID NO:590, has at least 99% identity to the sequence set forth in SEQ ID NO:590, having at least 98% identity to the sequence set forth in SEQ ID NO:590, having at least 97% identity to the sequence set forth in SEQ ID NO:590, having at least 96% identity to the sequence set forth in SEQ ID NO:590, which has at least 95% identity to the sequence given in SEQ ID NO:590, having at least 90% identity to the sequence set forth in SEQ ID NO:590 or a sequence having at least 85% identity to SEQ ID NO:590 to a sequence having at least 80% identity.

In one embodiment, the CCR of the present invention comprises a CD137 intracellular domain, also known as a 4-1BB intracellular domain, consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:591, or a sequence identical to SEQ ID NO:591 has at least 99% identity to the sequence given in SEQ ID NO:591 has at least 98% identity to the sequence given in SEQ ID NO:591 has at least 97% identity to the sequence given in SEQ ID NO:591 has at least 96% identity to the sequence given in SEQ ID NO:591 has at least 95% identity to the sequence given in SEQ ID NO:591 has at least 90% identity to the sequence given in SEQ ID NO:591 or a sequence having at least 85% identity to SEQ ID NO:591, a nucleotide code for a sequence having at least 80% identity to the sequence given.

In one embodiment, the CCR of the present invention comprises a CD27 intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:592, or a sequence that hybridizes to SEQ ID NO:592 has at least 99% identity to a sequence set forth in SEQ ID NO:592 has at least 98% identity to a sequence set forth in SEQ ID NO:592, having at least 97% identity to the sequence set forth in SEQ ID NO:592, having at least 96% identity to the sequence set forth in SEQ ID NO:592, having at least 95% identity to the sequence set forth in SEQ ID NO:592, having at least 90% identity to the sequence set forth in SEQ ID NO:592 or a sequence having at least 85% identity to SEQ ID NO:592 to a sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises a cd3ζ intracellular domain consisting of a polypeptide comprising SEQ ID NO:593, or a sequence identical to SEQ ID NO:593 has at least 99% identity to the sequence given in SEQ ID NO:593 has at least 98% identity to the sequence given in SEQ ID NO:593 has at least 97% identity to the sequence given in SEQ ID NO:593 has at least 96% identity to the sequence given in SEQ ID NO:593 has at least 95% identity to the sequence given in SEQ ID NO:593 has at least 90% identity to the sequence given in SEQ ID NO:593 or a sequence having at least 85% identity to SEQ ID NO:593 to seq id No. 3, a nucleotide code of a sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-2rβ intracellular domain consisting of a polypeptide comprising SEQ ID NO:594, or a sequence identical to SEQ ID NO:594, has at least 99% identity to the sequence set forth in SEQ ID NO:594, has at least 98% identity to the sequence set forth in SEQ ID NO:594, has at least 97% identity to the sequence set forth in SEQ ID NO:594, has at least 96% identity to the sequence set forth in SEQ ID NO:594, has at least 95% identity to the sequence set forth in SEQ ID NO:594, has at least 90% identity to the sequence set forth in SEQ ID NO:594 or a sequence having at least 85% identity to SEQ ID NO:594 to seq id no.

In one embodiment, a CCR of the invention comprises an IL-2rγ intracellular domain consisting of a polypeptide comprising SEQ ID NO:595, or a sequence identical to SEQ ID NO:595, has at least 99% identity to the sequence set forth in SEQ ID NO:595, has at least 98% identity to the sequence set forth in SEQ ID NO:595, has at least 97% identity to the sequence set forth in SEQ ID NO:595, has at least 96% identity to the sequence set forth in SEQ ID NO:595, has at least 95% identity to the sequence set forth in SEQ ID NO:595, has at least 90% identity to the sequence set forth in SEQ ID NO:595 or a sequence having at least 85% identity to SEQ ID NO:595, a nucleotide encoding a sequence having at least 80% identity to the sequence given.

In one embodiment, a CCR of the invention comprises an IL-18R1 intracellular domain consisting of a polypeptide comprising SEQ ID NO:596, or a sequence identical to SEQ ID NO:596 has at least 99% identity to the sequence given in SEQ ID NO:596 has at least 98% identity to the sequence given in SEQ ID NO:596 has at least 97% identity to the sequence given in SEQ ID NO:596 has at least 96% identity to the sequence given in SEQ ID NO:596 has at least 95% identity to the sequence given in SEQ ID NO:596 has at least 90% identity to the sequence given in SEQ ID NO:596 or a sequence having at least 85% identity to SEQ ID NO:596, a nucleotide code for a sequence having at least 80% identity to the sequence given.

In one embodiment, a CCR of the invention comprises an IL-7rα intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:597, or a sequence identical to SEQ ID NO:597 has at least 99% identity to the sequence given in SEQ ID NO:597 has at least 98% identity to the sequence given in SEQ ID NO:597 has at least 97% identity to the sequence given in SEQ ID NO:597 has at least 96% identity to the sequence given in SEQ ID NO:597 has at least 95% identity to the sequence given in SEQ ID NO:597 has at least 90% identity to the sequence given in SEQ ID NO:597 or a sequence having at least 85% identity to SEQ ID NO:597, a nucleotide code for a sequence having at least 80% identity to the sequence given.

In one embodiment, a CCR of the invention comprises an IL-12R1 intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:598, or a sequence identical to SEQ ID NO:598 has at least 99% identity to the sequence given in SEQ ID NO:598 has at least 98% identity to the sequence set forth in SEQ ID NO:598 has at least 97% identity to the sequence given in SEQ ID NO:598 has at least 96% identity to the sequence given in SEQ ID NO:598 has at least 95% identity to the sequence given in SEQ ID NO:598 has at least 90% identity to the sequence given in SEQ ID NO:598 or a sequence having at least 85% identity to SEQ ID NO:598, a nucleotide code for a sequence having at least 80% identity to the sequence given.

In one embodiment, a CCR of the invention comprises an IL-12R2 intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:599, or a sequence identical to SEQ ID NO:599 has at least 99% identity to the sequence given in SEQ ID NO:599 has at least 98% identity to the sequence given in SEQ ID NO:599 has at least 97% identity to the sequence given in SEQ ID NO:599 has at least 96% identity to the sequence given in SEQ ID NO:599 has at least 95% identity to the sequence given in SEQ ID NO:599 has at least 90% identity to the sequence given in SEQ ID NO:599 has at least 85% identity or is identical to the sequence given in SEQ ID NO:599 nucleotide codes for a sequence having at least 80% identity to the sequence given.

In one embodiment, a CCR of the invention comprises an IL-15 ra intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:600, or a sequence identical to SEQ ID NO:600, having at least 99% identity to the sequence set forth in SEQ ID NO:600, having at least 98% identity to SEQ ID NO:600, having at least 97% identity to the sequence set forth in SEQ ID NO:600, having at least 96% identity to the sequence set forth in SEQ ID NO:600, having at least 95% identity to the sequence set forth in SEQ ID NO:600, having at least 90% identity to the sequence set forth in SEQ ID NO:600 or a sequence having at least 85% identity to SEQ ID NO:600, and a sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-21R intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:601, or a sequence identical to SEQ ID NO:601 has at least 99% identity to the sequence set forth in SEQ ID NO:601 has at least 98% identity to the sequence set forth in SEQ ID NO:601 has at least 97% identity to the sequence given in SEQ ID NO:601 has at least 96% identity to the sequence given in SEQ ID NO:601 has at least 95% identity to the sequence set forth in SEQ ID NO:601 has at least 90% identity to the sequence set forth in SEQ ID NO:601 has at least 85% identity or NO:601 has a nucleotide code of a sequence having at least 80% identity.

In one embodiment, a CCR of the invention comprises an IL-21R intracellular domain consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:602, or a sequence identical to SEQ ID NO:602, has at least 99% identity to the sequence set forth in SEQ ID NO:602 has at least 98% identity to the sequence set forth in SEQ ID NO:602, has at least 97% identity to the sequence set forth in SEQ ID NO:602, has at least 96% identity to the sequence set forth in SEQ ID NO:602, has at least 95% identity to the sequence set forth in SEQ ID NO:602, has at least 90% identity to the sequence set forth in SEQ ID NO:602 has at least 85% identity or is identical to SEQ ID NO:602, a nucleotide code for a sequence having at least 80% identity to the sequence set forth herein.

In some embodiments, the CCR of the invention comprises an IL-21RAP (interleukin 18 receptor accessory protein) intracellular domain. The IL-18RAP domain is described in the following examples and is also disclosed in U.S. patent application publication No. US 2019/0350974 A1, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the CCR of the present invention comprises an IL-18RAP intracellular domain consisting of a polypeptide comprising the sequence set forth in U.S. patent application publication No. US 2019/0350974 A1: 10 or a nucleotide encoding a sequence having at least 99% identity to the sequence, at least 98% identity to the sequence, at least 97% identity to the sequence, at least 96% identity to the sequence, at least 95% identity to the sequence, at least 90% identity to the sequence, at least 85% identity to the sequence, or at least 80% identity to the sequence.

In some embodiments, the intracellular domain may be linked to the transmembrane domain by a linker consisting of a sequence comprising an SED ID NO: 603. In some embodiments, the intracellular domain can extend to include a short portion (e.g., 1 to 15 amino acids in length) of the transmembrane domain and the extracellular domain, which is then operably linked to the remainder of the extracellular binding domain.

In one embodiment, the CCR of the present invention comprises an intracellular or co-stimulatory domain comprising a STAT3 signaling domain. In one embodiment, a CCR of the present invention comprises an intracellular domain comprising a JAK-STAT pathway signaling domain. Suitable STAT3 and JAK-STAT domains are described in Kagoya et al, nature med.2018,24,352-359, and U.S. patent No. 10,822,392, the disclosures of each of which are incorporated herein by reference in their entirety.

In one embodiment, the CCR of the present invention comprises an intracellular or co-stimulatory domain comprising a CD40 ligand (CD 40L or CD 154) signaling domain. Suitable CD40L signaling domains are described in Aloui et al, int.J.mol.Sci.2014,15 (12), 22342-22364, and U.S. Pat. No. 10,287,354, the disclosures of each of which are incorporated herein by reference in their entirety.

D. Gene expression method

In some embodiments, the method of genetically modifying a TIL population to express CCR or chemokine receptors comprises the step of stably incorporating the gene to produce more than one protein. In one embodiment, the method of genetically modifying a TIL population comprises a viral transduction step. In one embodiment, the method of genetically modifying a population of TILs comprises a retroviral transduction step. In one embodiment, the method of genetically modifying a TIL population comprises a gamma retrovirus transduction step. In one embodiment, the method of genetically modifying a TIL population comprises an adenovirus transduction step. In one embodiment, the method of genetically modifying a TIL population comprises an adenovirus-associated transduction step. In one embodiment, the method of genetically modifying a TIL population comprises a herpes simplex virus transduction step. In one embodiment, the method of genetically modifying a population of TILs comprises a poxvirus transduction step. In some embodiments, the method of genetically modifying a population of TILs comprises a lentiviral transduction step comprising lentiviral transduction using a Human Immunodeficiency Virus (HIV), including HIV-1. Lentiviral transduction systems and other suitable viral transduction systems are known in the art and are described, for example, in Levine et al, proc.nat' l acad.sci.2006,103,17372-77; zufferey et al, nat. Biotechnol.1997,15,871-75; dull et al, J.virology 1998,72,8463-71 and U.S. Pat. No. 5,350,674; 5,585,362; and 6,627,442, the disclosures of each of which are incorporated herein by reference in their entirety. In one embodiment, the method of genetically modifying a TIL population comprises a gamma retrovirus transduction step. Gamma retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, cur. Prot. Mol. Biol.1996,9.9.1-9.9.16, hawley et al, gene Ther.1994,1,136-38; the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the pqxix retroviral vector is used to genetically modify a TIL population to express CCR or chemokine receptors of the invention. Other viral systems known in the art can be similarly employed to modify the TIL population to stably or transiently express CCR.

In some embodiments, the method of genetically modifying a TIL population to express CCR comprises the step of preparing a lentiviral vector derived from at least a portion of a lentiviral genome, including specifically a self-inactivating lentiviral vector, as provided by Milone et al, mol. Ter. 2009,17, 1453-1464. Other examples of lentiviral vectors that may be used clinically include, but are not limited to, LENTrVECTOR such as Oxford BioMedica ^TM LENTIMAX of Lentigen ^TM Carrier systems and the like. In some embodiments, the lentiviral vector carrying the transgene is combined with a vesicular stomatitis virus glycoprotein (VSV-G) plasmid that results in a ubiquitous phospholipid component with the serosa rather thanExpression of specific cell surface receptors and plasmid-bound rhabdovirus envelope proteins. In some embodiments, the lentiviral vector carrying the transgene is combined with Gag/Pol and Rev packaging plasmids. In some embodiments, lentiviral packaging is performed using a 293T cell line, such as a HEK293T cell line or variants, derivatives, or progeny thereof.

In one embodiment, the viral or lentiviral vector backbone is pgem.64a, as described in Zhao et al, mol. Ther.2006,13,151-9, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the lentiviral vector backbone is pFUGW, as described by Lois et al, science 2002,295,868-72, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the lentiviral vector comprises a bovine growth hormone poly-A sequence to drive transgene expression. In one embodiment, the lentiviral vector comprises a Kozak ribosome initiation sequence. In one embodiment, the lentiviral vector comprises a native murine hepatitis virus post-transcriptional regulatory element (WPRE). In one embodiment, the lentiviral vector comprises a Long Terminal Repeat (LTR) sequence derived from a pFUGW plasmid. In one embodiment, the viral or lentiviral vector backbone is pRRLSIN. In one embodiment, the viral or lentiviral vector backbone is pLenti. In one embodiment, the viral or retroviral vector backbone is pqxix.

In one embodiment, the viral or lentiviral vector backbone further comprises a promoter. In one embodiment, the promoter is a human elongation growth factor-1 or EF-1 promoter. In one embodiment, the promoter is an EF-1 alpha (also known as EF-1a or EF-1 alpha) promoter. In one embodiment, the promoter is SEQ ID NO:604 or a functional part or functional variant thereof. In one embodiment, the promoter is an immediate early Cytomegalovirus (CMV) promoter sequence, which is a strong constitutive promoter sequence capable of driving high expression of any polynucleotide sequence operably linked thereto. In one embodiment, the promoter is SEQ ID NO:605 or a functional portion or functional variant thereof. In one embodiment, the viral vector backbone further comprises a promoter, wherein the promoter is a murine embryonic stem cell virus (MSCV) promoter. In one embodiment, the promoter is SEQ ID NO:606 or a functional portion or functional variant thereof. In one embodiment, the viral or lentiviral vector backbone further comprises a promoter, wherein the promoter is an activated T cell Nuclear Factor (NFAT) promoter. Suitable NFAT promoters are described in U.S. patent No. 8,556,882 and Merlet et al, gene Therapy 2013,20,248-254, the disclosures of which are incorporated herein by reference in their entirety, and which may include more than one NFAT binding motif, including NFAT1, NFAT2, NFAT3, and NFAT4 reactive elements. In one embodiment, the NFAT promoter comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve binding motifs. In one embodiment, the NFAT promoter comprises up to twelve binding motifs. In one embodiment, the NFAT promoter comprises four, five, six, or seven binding motifs. In one embodiment, the NFAT promoter comprises six binding motifs. In one embodiment, the promoter is SEQ ID NO:607 or a functional portion or functional variant thereof.

In one embodiment, the promoter is selected from the group consisting of EF-1 promoter, simian virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) LTR promoter, MSCV promoter, NFAT promoter, moloney murine leukemia virus (MoMuLV) promoter, avian leukemia virus promoter, ai Baer immediate early promoter, rous sarcoma virus promoter, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the promoter is an inducible promoter, in some embodiments, which can initiate expression of its operably linked polynucleotide sequence when expression is desired, or shut down expression when expression is not desired. In one embodiment, the inducible promoter is selected from the group consisting of a metallothionein promoter, a glucocorticosteroid promoter, a progestin promoter, and a tetracycline promoter. Exemplary, non-limiting sequences of suitable promoters are provided in table 60.

Table 60: nucleotide sequence of exemplary promoters

In one embodiment, the promoter domain used in a CCR or chemokine receptor encoding vector for modifying a TIL, MILs, or PBL as described herein comprises SEQ ID NO: 604. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 99% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 98% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 97% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 96% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 95% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 90% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 85% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:604 has a nucleotide sequence having at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:604 is optimized to improve protein expression.

In one embodiment, the promoter domain used in a CCR or chemokine receptor encoding vector for modifying a TIL, MILs, or PBL as described herein comprises SEQ ID NO: 605. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 99% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 98% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 97% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 96% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 95% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 90% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 85% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:605 has a nucleotide sequence having at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:605 is optimized to improve protein expression.

In one embodiment, the promoter domain used in a CCR or chemokine receptor encoding vector for modifying a TIL, MILs, or PBL as described herein comprises SEQ ID NO: 606. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence of at least 99% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence of at least 98% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence of at least 97% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence of at least 96% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence of at least 95% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence that is at least 90% identical. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence of at least 85% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:606 has a nucleotide sequence of at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:606 is optimized to improve protein expression.

In one embodiment, the promoter domain used in a CCR or chemokine receptor encoding vector for modifying a TIL, MILs, or PBL as described herein comprises SEQ ID NO:607, and a nucleotide sequence of seq id no. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 99% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 98% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 97% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 96% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 95% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 90% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 85% identity. In one embodiment, the promoter domain used in a vector encoding a CCR or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:607 has a nucleotide sequence of at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:607 is optimized to improve protein expression.

In one embodiment, the vector is an in vitro transcription vector, including a vector that transcribes the RNA of the nucleic acid molecules described herein. In one embodiment, the nucleic acid sequence of the vector further comprises a polyadenylation or poly (a) tail comprising about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 adenosine bases. In one embodiment, the nucleic acid sequence in the vector further comprises a 3 'untranslated region (UTR) comprising at least one repeat of a 3' UTR derived from a human β -globulin.

The invention also includes RNA constructs that can be transfected directly into cells. One method for generating mRNA for transfection involves In Vitro Transcription (IVT) of a template with specially designed primers and subsequent addition of poly (A) to produce a construct of typically 50 to 2000 bases in length containing 3' and 5' UTRs, 5' covers and/or Internal Ribosome Entry Sites (IRES), nucleic acid to be expressed and a polyadenylation tail. The RNA thus produced can be used to efficiently transfect different kinds of cells. In one embodiment, the template comprises the sequence of CCR as described herein. In one embodiment, the RNA CCR vector is transduced into T cells by electroporation.

In one embodiment, suitable transgenic substituted vector expression systems disclosed in U.S. patent application publication No. US 2019/0298770 A1 and encoding CCR of the present invention may be used, the disclosure of these vector expression systems being incorporated herein by reference in their entirety.

In one embodiment, a method of genetically modifying a population of TILs comprises the step of transposon mediated gene transfer. Transposon mediated gene transfer systems are known in the art and include systems in which a 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, e.g., the transposase is provided as mRNA (e.g., mRNA comprising a cap and a polyadenylation tail). Suitable transposon mediated gene transfer systems include salmon-type Tel-like translocases (SB or sleeping beauty translocases) such as SB10, SB11 and SB100x, and engineered enzymes with increased enzymatic activity are described, for example, in Hackett et al, mol. Therapy 2010,18,674-83 and U.S. patent No. 6,489,458, the respective disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, CCR is transiently expressed by the TIL population. In some embodiments, CCR is transiently expressed by RNA electroporation through the TIL population. In one embodiment, the method of genetically modifying a population of TILs comprises an electroporation step. Electroporation methods are known in the art and are described, for example, in Tsong, biophys.j.1991,60,297-306 and U.S. patent application publication No. 2014/0227237 A1, the respective disclosures of which are incorporated herein by reference in their entirety. Other electroporation methods known in the art may be used, such as described in 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 their entirety. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, 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 a defined and controlled permanent or temporary change in the TIL, comprising the step of applying at least three single, operator controlled, independently programmed sequences of DC electrical pulses to the TIL having a field strength equal to or greater than 100V/cm, wherein the at least three sequences of DC electrical pulses have 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) the second first pulse interval in the first set of at least three pulses is different from the second pulse interval in the second set of at least three pulses. In one embodiment, 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 at least three single, operator controlled, independently programmed sequences of DC electrical pulses to the TIL having a field strength equal to or greater than 100V/cm, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In one embodiment, 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 at least three single, operator controlled, independently programmed sequences of DC electrical pulses to the TIL having a field strength equal to or greater than 100V/cm, wherein at least two of the at least three pulses differ from each other in pulse width. In one embodiment, 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 a defined and controlled permanent or temporary change in the TIL, comprising the step of applying at least three single, operator controlled, independently programmed sequences of DC electrical pulses having a field strength equal to or greater than 100V/cm to the TIL, wherein a first pulse interval of a second of the first set of at least three pulses is different from a second pulse interval of a second of the second set of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method comprising the step of treating a TIL with a pulsed electric field to induce void formation in the TIL, comprising the step of applying a sequence of at least three DC electrical pulses having a field strength equal to or greater than 100V/cm to the TIL, wherein the sequence of at least three DC electrical 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) the first pulse interval of the second of the first set of at least three pulses is different from the second pulse interval of the second set of at least three pulses such that the induced holes last for a relatively long period of time and such that the viability of the TIL is maintained. In one embodiment, the method of genetically modifying a population of TILs comprises a calcium phosphate transfection step. The calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in Graham and van der Eb, virology 1973,52,456-467; wigler et al, proc.Natl.Acad.Sci.1979,76,1373-1376, chen and Okayarea, 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 their entirety. In one embodiment, the method of genetically modifying a population of TILs comprises a liposome transfection step. Liposome transfection methods such as 1 using the cationic lipids N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) and dioleylphospholipid ethanolamine (DOPE) in filtered water: methods of 1 (w/w) liposome formulation are known in the art and are described in Rose et al, biotechniques 1991,10,520-525 and Felgner et al, proc. Natl. Acad. Sci. USA,1987,84,7413-7417 and U.S. Pat. No. 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 their entirety. In one embodiment, a method of genetically modifying a population of TILs comprises using us patent 5,766,902;6,025,337;6,410,517; a transfection step of the methods described in 6,475,994 and 7,189,705; the respective disclosures of which are incorporated herein by reference in their entirety.

According to one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises:

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

(b) Adding the tumor fragments to a closed system;

(c) Producing a second population of TILs by culturing the 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 from the start of the amplification process) for a first amplification, wherein the first amplification is performed in a closed container providing a first gas-permeable surface area, the first amplification is performed for about 3 to 14 days to obtain the second population of TILs, the transition from step (b) to step (c) occurs without opening the system;

(d) Generating a third population of TILs by supplementing or replacing cell culture media of the second population of TILs with additional IL-2, culture media, optionally OKT-3, and Antigen Presenting Cells (APCs) for a second amplification, the second amplification being 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) occurring without opening the system;

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

(f) Transferring the collected population of TILs from step (e) to an infusion bag, wherein the transfer from step (e) to step (f) occurs without opening the system; and

(g) At any time from step (e) to step (f), at least a portion of the TIL is genetically modified to stabilize or transiently express one or more chimeric co-stimulatory receptors.

As described in step (g) of the above embodiments, the genetic modification process may be performed at any time during the TIL amplification method, in other words, the genetic modification may be performed at the TIL before, during or after any step of the amplification method; for example, during or before or after any of steps (a) to (f) of the method overview above. According to certain embodiments, the TILs are collected during the amplification method (e.g., the amplification method is "paused" for at least a portion of the TILs) and the collected TILs are subjected to a genetic modification process, in some instances followed by reintroduction back into the amplification method (e.g., back into the culture medium) to continue the amplification process such that at least a portion of the therapeutic TIL population eventually transferred to the infusion bag is permanently gene edited. In one embodiment, the genetic modification process may be performed prior to amplification by activating the TIL, performing a genetic modification step on the activated TIL, and amplifying the genetically modified TIL according to the processes described herein. In any of the foregoing embodiments, the genetic modification process can result in expression of CCR as described herein. It should be noted that alternative embodiments of the amplification process may differ from the methods shown above. Alternative embodiments may not have the same steps (a) to (g) or 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 rapid amplifications, and gene editing may be performed at the TIL during the third or fourth amplifications.

In some embodiments, the gene editing process is performed on more than one TIL from the first population, the second population, and the third population of the TIL manufacturing process described herein. For example, gene editing may be performed on the first population of TILs or on a portion of TILs collected from the first population, after which the TILs may be subsequently placed back into the amplification process (e.g., back into the culture medium). Alternatively, gene editing may be performed separately from the TIL from the second or third population or from a portion of the TIL collected from the second or third population, after which the TIL may be subsequently placed back into the amplification process (e.g., back into the culture medium). According to another embodiment, the gene editing is performed while the TIL is maintained in the medium and while the amplification is ongoing.

In some embodiments, the gene editing process is performed on TIL from the first amplification or TIL from the second amplification or TIL from both the first and second amplifications. For example, during the first amplification or the second amplification, gene editing may be performed on the TIL collected from the culture medium, after which the TIL may be subsequently returned to the amplification method, e.g., reintroduced back into the culture medium.

In some embodiments, the gene editing process is performed on at least a portion of the TIL after the first amplification and before the second amplification. For example, after a first amplification, gene editing may be performed on the TIL collected from the medium, after which the TIL may be subsequently returned to the amplification method, e.g., reintroduced back into the medium of a second amplification.

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

In some embodiments, the cell culture medium may comprise OKT-3 from the start day of first expansion (day 0) or from day 1, such that gene editing occurs after TIL is exposed to OKT-3 on day 0 and/or day 1 in the cell culture medium. In some embodiments, the cell culture medium during the first expansion and/or during the second expansion comprises OKT-3 and the gene editing is performed prior to the introduction of OKT-3 into the cell culture medium. Alternatively, the cell culture medium during the first expansion and/or during the second expansion may comprise OKT-3 and the gene editing is performed after the OKT-3 is introduced into the cell culture medium.

In some embodiments, the cell culture medium may comprise a 4-1BB agonist or OX40 agonist from the start of the first expansion day (day 0) or from day 1, such that gene editing occurs after TIL is exposed to the 4-1BB agonist or OX40 agonist on day 0 and/or day 1 in the cell culture medium. In some embodiments, the cell culture medium during the first expansion and/or during the second expansion comprises a 4-1BB agonist or an OX40 agonist and gene editing is performed prior to introduction of the 4-1BB agonist or the OX40 agonist into the cell culture medium. In some embodiments, the cell culture medium during the first expansion and/or during the second expansion may comprise a 4-1BB agonist or an OX40 agonist and gene editing is performed after the 4-1BB agonist or the OX40 agonist is introduced into the cell culture medium.

In some embodiments, the cell culture medium may comprise IL-2 from the start day of first expansion (day 0) or from day 1, such that gene editing occurs after IL exposure to IL-2 in the cell culture medium on day 0 and/or day 1. According to another embodiment, the cell culture medium during the first expansion and/or during the second expansion comprises IL-2 and the gene editing is performed before the IL-2 is introduced into the cell culture medium. Alternatively, the cell culture medium during the first expansion and/or during the second expansion may comprise IL-2 and the gene editing is performed after the IL-2 is introduced into the cell culture medium.

As discussed above, one or more of the OKT-3, 4-1BB agonist and IL-2 may be included in the cell culture medium starting on day 0 or day 1 of the first amplification. According to one embodiment, OKT-3 is included in the cell culture medium from day 0 or day 1 of the first amplification, and/or the 4-1BB agonist is included in the cell culture medium from day 0 or day 1 of the first amplification, and/or IL-2 is included in the cell culture medium from day 0 or day 1 of the first amplification. According to one example, the cell culture medium comprises OKT-3 and 4-1BB agonists starting on day 0 or day 1 of the first expansion. According to another example, 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 additional point in time during the amplification process, as shown in the various embodiments described herein.

(a) Obtaining a first TIL population from a resected tumor of a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments or tumor digests, optionally after thawing the cryopreserved plurality of tumor fragments or cryopreserved tumor digests;

(b) Adding tumor fragments or tumor digests to a closed system;

(c) Performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) Adding OKT-3 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 1 to 3 days to obtain a second population of TILs, the second population of TILs being at least 50 times higher in number than the first population of TILs, the transition from step (c) to step (d) occurring without opening the system;

(e) Optionally genetically modifying at least a portion of the TIL to stabilize or transiently express one or more chimeric co-stimulatory receptors by transferring the portion of the TIL to a temporary container;

(f) Optionally allowing the second TIL population to rest for about 1 day to about 5 days;

(g) Performing a second amplification by supplementing cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and Antigen Presenting Cells (APC) to produce a third TIL population, wherein the second amplification is performed for about 7 to 11 days to obtain the third TIL population, 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) occurs without opening the system;

(h) Collecting the therapeutic TIL population obtained from step (g) to provide 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;

(i) Transferring the collected TIL population to an infusion bag, wherein the transfer from step (h) to step (i) occurs without opening the system; and

(j) The collected TIL populations were cryopreserved using a dimethylsulfoxide-based cryopreservation medium.

According to one embodiment, the foregoing methods may be used to provide a population of collected autologous TILs to treat a human cancer subject.

In some embodiments, the vector encoding CCR and/or chemokine receptors for modifying a TIL, MILs, or PBL as described herein comprises a nucleotide sequence encoding a 2A self-cleaving peptide. The CCR and chemokines of the invention may employ vectors encoding 2A self-cleaving peptides to induce ribosome jump during translation and production of multimeric proteins (ribosomal skipping). Without being bound by theory, a vector encoding a CCR or chemokine receptor comprising a 2A self-cleaving peptide may be cleaved into two proteins, e.g., two CCR or CCR and another protein, upon intracellular expression. The amino acid sequences of exemplary and non-limiting 2A self-cleaving peptide domains are provided in table 61. SEQ ID NO:608 is the amino acid sequence of the T2A self-cleaving peptide (derived from the vein occlusion virus 2A), SEQ ID NO:609 is the amino acid sequence of the P2A self-cleaving peptide (derived from porcine teschovirus-1 2A), SEQ ID NO:610 is the amino acid sequence of the E2A self-cleaving peptide (derived from equine rhinitis a virus) and SEQ ID NO:611 is the amino acid sequence of the F2A self-cleaving peptide (derived from foot and mouth disease virus).

Table 61: exemplary amino acid sequence of 2A self-cleaving peptide

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO:608, and a sequence of amino acids. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 95% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 90% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:608 has an amino acid sequence having at least 80% identity.

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO: 609. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 95% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 90% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:609 has an amino acid sequence having at least 80% identity.

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO: 610. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence having at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence having at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence having at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence having at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence that is at least 95% identical. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence having at least 90% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence having at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:610 has an amino acid sequence having at least 80% identity.

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO: 611. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 95% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 90% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:611 has an amino acid sequence having at least 80% identity.

The aforementioned 2A self-cleaving peptide domain may also be combined at the N-terminus with a linker such as GSG linker (SEQ ID NO: 612). Alternative linkers include SEQ ID NOs: 238. SEQ ID NO: 239. SEQ ID NO: 240. SEQ ID NO: 241. SEQ ID NO: 242. SEQ ID NO: 243. SEQ ID NO: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 70. SEQ ID NO: 71. SEQ ID NO: 72. SEQ ID NO: 74. conservative amino acid substitutions thereof, variants thereof, or other linkers known in the art, including those described by Bird et al, science 1988,242,423-426, the disclosure of which is incorporated herein by reference in its entirety.

Exemplary, non-limiting nucleotide sequences for suitable 2A self-cleaving peptide domains are provided in table 62.

Table 62: exemplary nucleotide sequence of 2A self-cleaving peptide

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO: 613. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has a nucleotide sequence of at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has a nucleotide sequence of at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has a nucleotide sequence of at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has a nucleotide sequence of at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has a nucleotide sequence of at least 95% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has a nucleotide sequence of at least 90% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has a nucleotide sequence of at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:613 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:613 is optimized to improve protein expression.

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO: 614. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 95% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 90% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:614 has a nucleotide sequence of at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:614 are optimized to improve protein expression.

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO: 615. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence of at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence of at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence of at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence of at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence of at least 95% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence that is at least 90% identical. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence of at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:615 has a nucleotide sequence of at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:615 is optimized to improve protein expression.

In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises SEQ ID NO: 616. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 99% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 98% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 97% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 96% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 95% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 90% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 85% identity. In one embodiment, the 2A self-cleaving peptide domain used to modify a vector encoding a CCR and/or chemokine receptor of a TIL, MIL or PBL as described herein comprises a sequence identical to SEQ ID NO:616 has a nucleotide sequence of at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:616 is optimized to improve protein expression.

In one embodiment, the vector encoding the CCR and/or chemokine receptor of the invention comprises an IRES domain. Suitable IRES domains are known in the art. In one embodiment, the vector encoding the CCR and/or chemokine receptor of the invention comprises the amino acid sequence of SEQ ID NO: 617. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 99% identity. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 98% identity. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 97% identity. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 96% identity. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 95% identity. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 90% identity. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 85% identity. In one embodiment, a vector encoding a CCR and/or chemokine receptor for modifying a TIL, MILs, or PBL as described herein comprises a sequence identical to SEQ ID NO:617 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:617 is optimized to improve protein expression. In embodiments including the preceding embodiments, SEQ ID NO:617 is optimized to improve protein expression. Other suitable IRES domains are described in Bochkov and Palmen berg, biotechniques 2006,41 (3), 283, the disclosure of which is incorporated herein by reference in its entirety.

CCR construct

The aforementioned extracellular and intracellular domains may be combined and optionally the transmembrane domains further combined to provide a CCR suitable for the TIL of the present invention. Several exemplary CCR constructs of the invention have been previously discussed, and fig. 37 depicts or describes herein other exemplary CCR constructs, each of which is an embodiment of the invention.

In some embodiments, a CCR of the invention comprises an extracellular domain selected from the group consisting of a PD-1 domain, a FAS binding domain, a tgfβ binding domain, a PD-L1 scFv binding domain, a CEA scFv binding domain, a CD73 scFv binding domain, a TROP-2 scFv binding domain, an EPCAM scFv binding domain, a tissue factor scFv binding domain, a FAP scFv binding domain, a LFA-1 scFv binding domain, a VISTA scFv binding domain, and a LLRC15 scFv binding domain.

In some embodiments, a CCR of the invention comprises an extracellular domain that binds to a molecule selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, fra, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, FAS, tgfβ, tgfβrii, and MUC 16.

In some embodiments, the CCR of the present invention comprises: (i) An extracellular domain selected from the group consisting of a PD-1 domain, a PD-L1 scFv binding domain, a CEA scFv binding domain, a CD73 scFv binding domain, a TROP-2 scFv binding domain, an EPCAM scFv binding domain, a tissue factor scFv binding domain, an LFA-1 scFv binding domain, a FAP scFv binding domain, a VISTA scFv binding domain, and a LLRC15 scFv binding domain, and (ii) an intracellular domain selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, IL-2Rβ, IL-2Rγ, IL-18R1, IL-18RAP, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, and IL-21R.

In some embodiments, the CCR of the present invention comprises: (i) An extracellular domain that binds to a molecule selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, fra, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, and MUC16, and (ii) an intracellular domain selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, IL-2rβ, IL-2rγ, IL-18R1, IL-18RAP, IL-7rα, IL-12R1, IL-12R2, IL-15rα, and IL-21R.

In some embodiments, a CCR of the present invention is a protein comprising: (i) An extracellular domain selected from the group consisting of a PD-1 domain, a PD-L1 scFv binding domain, a CEA scFv binding domain, a CD73 scFv binding domain, a TROP-2 scFv binding domain, an EPCAM scFv binding domain, a tissue factor scFv binding domain, an LFA-1 scFv binding domain, a FAP scFv binding domain, a VISTA scFv binding domain, and a LLRC15 scFv binding domain, and (ii) an intracellular domain selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2rβ domain, an IL-2rγ domain, an IL-18R1 domain, an IL-7rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15rα domain, an IL-21R domain, and combinations thereof. In some embodiments, the extracellular domain and the intracellular domain are operably linked. In some embodiments, the extracellular domain and the intracellular domain are connected by a linker domain.

In some embodiments, a CCR of the present invention is a protein comprising: (i) an extracellular domain selected from the group consisting of a PD-1 domain, a PD-L1 scFv binding domain, a CEA scFv binding domain, a CD73 scFv binding domain, a TROP-2 scFv binding domain, an EPCAM scFv binding domain, a tissue factor scFv binding domain, an LFA-1 scFv binding domain, a FAP scFv binding domain, a VISTA scFv binding domain, and a LLRC15 scFv binding domain, (ii) a transmembrane domain selected from the group consisting of a CD3 alpha domain, a CD3 beta domain, a CD zeta domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 alpha domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, a IgG4 domain, an IL-18 domain, an IL-2R 2 alpha domain, and an IL-2 beta domain, and (iii) an intracellular domain selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2Rβ domain, an IL-2Rγ domain, an IL-18R1 domain, an IL-18RAP domain, an IL-7Rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15Rα domain, an IL-21R domain, and combinations thereof. In some embodiments, the extracellular domain and the transmembrane domain are operably linked, and the transmembrane domain and the intracellular domain are operably linked. In some embodiments, the extracellular domain, the transmembrane domain, and the intracellular domain are each interconnected by a linker domain.

In some embodiments, a CCR of the present invention is a protein comprising: (i) an extracellular domain selected from the group consisting of a PD-1 domain, a PD-L1 scFv binding domain, a CEA scFv binding domain, a CD73 scFv binding domain, a TROP-2 scFv binding domain, an EPCAM scFv binding domain, a tissue factor scFv binding domain, an LFA-1 scFv binding domain, a FAP scFv binding domain, a VISTA scFv binding domain, and a LLRC15 scFv binding domain, (ii) a transmembrane domain selected from the group consisting of a CD3 alpha domain, a CD3 beta domain, a CD zeta domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 alpha domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, a 4 domain, an IL-2R alpha domain, an IL-2 beta domain, and an IL-R2 gamma domain, (iii) a hinge protein domain selected from the group consisting of a CD3 a domain, a CD3 β domain, a cdζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 a domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2 ra domain, an IL-2rβ domain, and an IL-2rγ domain, and (iv) an intracellular domain selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, CD137 (4-1 BB) domain, CD27 domain, IL-2Rβ domain, IL-2Rγ domain, IL-18R1 domain, IL-18RAP domain, IL-7Rα domain, IL-12R1 domain, IL-12R2 domain, IL-15Rα domain, IL-21R domain, and combinations thereof. In some embodiments, the extracellular domain and the hinge domain are operably linked, the hinge domain and the transmembrane domain are operably linked, and the transmembrane domain and the intracellular domain are operably linked. In some embodiments, the extracellular domain, the transmembrane domain, and the intracellular domain are each interconnected by a linker domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a PD-1 domain and a PD-1 transmembrane domain. In one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence of amino acids operably linked to SEQ ID NO:245, SEQ ID NO:244. in one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence linked to SEQ ID NO:245, SEQ ID NO:244. in one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence of amino acids operably linked to SEQ ID NO:245, SEQ ID NO:244 further operably linked to an intracellular domain. In one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence of amino acids operably linked to SEQ ID NO:245, SEQ ID NO:244 further operably linked to an intracellular domain selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2rβ domain, an IL-2rγ domain, an IL-18R1 domain, an IL-7rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15rα domain, an IL-21R domain, and combinations thereof.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a PD-1 domain and a CD28 transmembrane domain. In one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence of amino acids operably linked to SEQ ID NO:246 of SEQ ID NO:244. in one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence linked to SEQ ID NO:246 of SEQ ID NO:244. in one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence of amino acids operably linked to SEQ ID NO:246 of SEQ ID NO:244 further operably linked to an intracellular domain. In one embodiment, the invention includes a TIL, MILs, or PBL expressing a protein sequence comprising a sequence of amino acids operably linked to SEQ ID NO:246 of SEQ ID NO:244 further operably linked to an intracellular domain selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2rβ domain, an IL-2rγ domain, an IL-18R1 domain, an IL-7rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15rα domain, an IL-21R domain, and combinations thereof.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., the domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., the domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., the domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., the domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., the domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-VISTA) domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a PD-L1 binding (anti-PD-L1) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CEA binding (anti-CEA) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a CD73 binding (anti-CD 73) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., the domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a TROP-2 binding (anti-TROP-2) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an EPCAM binding (anti-EPCAM) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a tissue factor binding (anti-TF) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LFA-1 binding (anti-LFA-1) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a FAP binding (anti-FAP) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., the domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., the domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising a VISTA binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a hinge domain, a transmembrane domain, and a CD28 intracellular domain (e.g., the domain of SEQ ID NO: 572).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a hinge domain, a transmembrane domain, and a CD134 (OX 40) intracellular domain (e.g., domain of SEQ ID NO: 573).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a hinge domain, a transmembrane domain, and a CD134 (ICOS) intracellular domain (e.g., domain of SEQ ID NO: 574).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-VISTA) domain, a hinge domain, a transmembrane domain, and a CD137 (4-1 BB) intracellular domain (e.g., the domain of SEQ ID NO: 575).

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a hinge domain, a transmembrane domain, and a CD27 intracellular domain (e.g., domain of SEQ ID NO: 576).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a hinge domain, a transmembrane domain, and an IL-2Rβ intracellular domain (e.g., the domain of SEQ ID NO: 578).

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an LRRC15 binding (anti-LRRC 15) domain, a hinge domain, a transmembrane domain, and an IL-18R1 intracellular domain (e.g., the domain of SEQ ID NO: 580).

In some embodiments, the invention includes TIL, MILs, or PBL expressing CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, optionally a CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes TIL, MILs, or PBL expressing CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, optionally a CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes TIL, MILs, or PBL expressing CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, optionally a CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes TIL, MILs, or PBL expressing CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, optionally a CD8 a hinge domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2Rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, an optional CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes TIL, MILs, or PBL expressing CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, optionally a CD8 a hinge domain, a CD28 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2Rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2Rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-B7-H3 scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAS scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tgfβrii scFv binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a PD1 binding domain, an IgG4 hinge domain, an IgG4 transmembrane domain, and an IL-2rβ intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-B7-H3 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAS binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tgfbetarii binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a PD1 binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and an IL-18R1 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-B7-H3 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAS scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tgfbetarii scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a PD-1 binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a CD27 intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-PD-L1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CEA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-CD 73 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-TROP-2 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-EPCAM scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tissue factor scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LFA-1 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAP scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-VISTA scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-LRRC 15 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MIL, or PBL that expresses a CCR comprising a protein sequence comprising an anti-B7-H3 scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-FAS scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising an anti-tgfbetarii scFv binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein sequence comprising a PD-1 binding domain, a CD8 a hinge domain, a CD8 a transmembrane domain, and a 4-1BB intracellular domain.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (CD 8 a hinge and transmembrane) - (IL-2 rβ), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (CD 8 a hinge and transmembrane) - (IL-18R 1), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 27), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 28), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 137), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a proteinThe protein comprises (anti-TROP-2-V) _L ) - (linker) - (anti-TROP-2-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 134), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 278), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (IgG 4 hinge and transmembrane) - (IL-2 rβ), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (IgG 4 hinge and transmembrane) - (IL-18R 1), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 27), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 28), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 137), wherein the domains are operably linked in brackets.

In some implementationsIn one embodiment, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 134), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-TROP-2-V _L ) - (linker) - (anti-TROP-2-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 278), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (CD 8 a hinge and transmembrane) - (IL-18R 1), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (CD 8 a hinge and transmembrane) - (IL-2 rβ), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (CD 8 a hinge and transmembrane) - (CD 27), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (CD 8. Alpha. Hinge and transmembrane) - (CD 28) wherein the domains are operably linked in brackets。

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (CD 8 a hinge and transmembrane) - (CD 137), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (CD 8 a hinge and transmembrane) - (CD 134), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (CD 8 a hinge and transmembrane) - (CD 278), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (IgG 4 hinge and transmembrane) - (IL-2 rβ), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (IgG 4 hinge and transmembrane) - (IL-18R 1), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (IgG 4 hinge and transmembrane) - (CD 27), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (IgG 4 hinge and transmembrane) - (CD 28), wherein the brackets indicateIs operably linked.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (IgG 4 hinge and transmembrane) - (CD 137), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (IgG 4 hinge and transmembrane) - (CD 134), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (anti-FAP-V _L ) - (linker) - (anti-FAP-V _H ) - (IgG 4 hinge and transmembrane) - (CD 278), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 a hinge and transmembrane) - (IL-18R 1), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 a hinge and transmembrane) - (IL-2 rβ), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H )-(CD 8 a hinge and transmembrane) - (CD 27), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 28), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 137), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 134), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 a hinge and transmembrane) - (CD 278), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (IgG 4 hinge and transmembrane) - (IL-2 rβ), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (IgG 4 hinge and transmembrane) - (IL-18R 1), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein thatComprises (anti-PD-L1-V) _L ) - (linker) - (anti-PD-L1-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 27), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 28), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 137), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 134), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (IgG 4 hinge and transmembrane) - (CD 278), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8a hinge and transmembrane) - (CD 27) using 38A1 anti-PD-L1V as described herein _H And V _L Domains, wherein each domain represented in brackets is operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8a hinge and transmembrane) - (CD 27) using 19H9 anti-PD-L1V as described herein _H And V _L Structure of theDomains, wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (hinge and transmembrane) - (4-1 BB intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (CD 8 signal peptide) - (anti-PD-L1-V) _L ) - (linker) - (anti-PD-L1-V) _H ) - (hinge and transmembrane) - (4-1 BB intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 hinge and transmembrane) - (4-1 BB intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (CD 8 signal peptide) - (anti-PD-L1-V) _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 hinge and transmembrane) - (4-1 BB intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (hinge and transmembrane) - (LTBR intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (CD 8 signal peptide) - (anti-PD-L1-V) _L ) - (linker) - (anti-PD-L1-V) _H ) - (hinge and transmembrane) - (LTBR intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 hinge and transmembrane) - (LTBR intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (CD 8 signal peptide) - (anti-PD-L1-V) _L ) - (linker) - (anti-PD-L1-V) _H ) - (CD 8 hinge and transmembrane) - (LTBR intracellular domain) optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (anti-PD-L1-V _L ) - (linker) - (anti-PD-L1-V) _H ) - (hinge and transmembrane) - (4-1 BB intracellular domain) - (LTBR intracellular domain) using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MIL or PBL that expresses a CCR comprising a protein comprising (CD 8 signal peptide) - (anti-PD-L1-V) _L ) - (linker) - (anti-PD-L1-V) _H ) - (hinge and transmembrane) - (LTBR intracellular domain) - (4-1 BB intracellular domain), optionally using 19H9 anti-PD-L1V as described herein _H And V _L Domains, each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (PD-1 ectodomain) - (12 amino acids of CD28 ectodomain) - (CD 28 transmembrane domain) - (CD 28 intracellular domain), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL expressing a CCR comprising a protein comprising (12 amino acids of the PD-1 ectodomain) - (4-1 BB transmembrane domain) - (4-1 BB intracellular domain), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (tgfbetarii ectodomain) - (12 amino acids of CD28 ectodomain) - (CD 28 transmembrane domain) - (CD 28 intracellular domain), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (tgfbetarii ectodomain) - (12 amino acids of 4-1BB ectodomain) - (4-1 BB transmembrane domain) - (4-1 BB intracellular domain), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL that expresses a CCR comprising a protein comprising (FAS ectodomain) - (FAS transmembrane domain) - (7 amino acids of FAS intracellular domain) - (4-1 BB intracellular domain), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes TIL, MILs, or PBL expressing CCR comprising a protein comprising (IgE signal peptide) - (PD-1 ectodomain) - (12 amino acids of CD28 ectodomain) - (CD 28 transmembrane domain) - (CD 28 intracellular domain), each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MILs, or PBL expressing a CCR comprising a protein comprising (IgE signal peptide) - (PD-1 ectodomain) - (12 amino acids of 4-1BB ectodomain) - (4-1 BB transmembrane domain) - (4-1 BB intracellular domain), the domains being operably linked in brackets.

In some embodiments, the invention includes TIL, MILs, or PBL expressing CCR comprising a protein comprising (IgE signal peptide) - (tgfbetarii ectodomain) - (12 amino acids of CD28 ectodomain) - (CD 28 transmembrane domain) - (CD 28 intracellular domain), each domain in brackets being operably linked.

In some embodiments, the invention includes a TIL, MILs, or PBL expressing a CCR comprising a protein comprising (IgE signal peptide) - (tgfbetarii ectodomain) - (12 amino acids of 4-1BB ectodomain) - (4-1 BB transmembrane domain) - (4-1 BB intracellular domain), the domains being operably linked in brackets.

In some embodiments, the invention includes a TIL, MILs, or PBL expressing a CCR comprising a protein comprising (IgE signal peptide) - (FAS extracellular domain) - (FAS transmembrane domain) - (7 amino acids of FAS intracellular domain) - (4-1 BB intracellular domain), wherein the domains are operably linked in brackets.

In some embodiments, the invention includes TIL, MILs, or PBL expressing a bi-epitope CCR construct.

The nucleotide sequences of the vectors encoding the exemplary CCR of the present invention are provided in table 63. In one embodiment, the nucleotide sequences in table 63 are codon optimized to improve protein expression. In one embodiment, the nucleotide sequence in table 63 is further modified to include additional linker domains as described elsewhere herein. In one embodiment, the nucleotide sequences in table 63 are used in a lentiviral expression system. In one embodiment, the nucleotide sequences in table 63 are used in a lentiviral expression system using an additional plasmid.

Exemplary carrier designs for the carriers provided in table 63 are provided in fig. 38-40. In one embodiment, the CCR encoded by the vector shown in fig. 38 is used to genetically modify the TIL products of the invention as described herein. In one embodiment, the CCR encoded by the vector shown in fig. 39 is used to genetically modify the TIL products of the invention as described herein. In one embodiment, the CCR encoded by the vector shown in fig. 40 is used to genetically modify the TIL products of the invention as described herein.

Table 63: nucleotide sequences of exemplary vectors for expression of CCR

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In one embodiment, the CCR comprises an anti-TROP-2-V _L -linker-anti-TROP-2-V _H -IgG4 (hinge and transmembrane) -IL2rβ construct. In one embodiment, CCR consists of a sequence comprising SEQ ID NO:618, and a nucleotide sequence encoding a nucleotide sequence of 618. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 99% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 98% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 97% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 96% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 95% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 94% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 93% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 92% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 91% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 have at least 90% identity The nucleotide sequence of the sex region encodes. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 85% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:618 having at least 80% identity.

In one embodiment, the CCR comprises an anti-FAP-V _L -linker-anti-FAP-V _H -CD8 a (hinge and transmembrane) -IL-18R1 construct. In one embodiment, CCR consists of a sequence comprising SEQ ID NO: 619. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 99% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 98% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 97% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 96% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 95% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 94% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 93% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 92% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 91% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 90% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 85% identity of the region of the nucleotide sequence code. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:619 has at least 80% identity of the region of the nucleotide sequence code.

In one embodiment, the CCR comprises, based on the 38A1 antibodies described hereinanti-PD-L1-V _L -linker-anti-PD-L1-V _H -CD 8a (hinge and transmembrane) -CD27 construct. In one embodiment, CCR consists of a sequence comprising SEQ ID NO:620, and the nucleotide sequence encoding the same. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 99% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 98% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 97% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 96% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 95% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 94% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 93% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 92% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 91% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 90% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 85% identity. In one embodiment, CCR consists of a sequence comprising a sequence identical to SEQ ID NO:620 having a region of at least 80% identity.

In one embodiment, using both the 38A1 and 19h9 PD-L1 domains described herein, the bi-epitope CCR comprises two CCR comprising SP- (38 A1 scFv) - (CD 28 hinge and transmembrane) - (IL-2 rβ intracellular) -T2A-SP- (19 h9 scFv) - (CD 28 hinge and transmembrane) - (IL-2 rγ intracellular), wherein SP refers to a signal peptide. In one embodiment, the bi-epitope CCR consists of a sequence comprising SEQ ID NO: 621. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 99% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 98% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 97% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 96% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 95% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 94% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 93% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 92% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 91% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 90% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 85% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:621 a region of at least 80% identity. The foregoing embodiments may include or omit fluorescent proteins, such as eGFP, as needed for analytical purposes.

In one embodiment, using both the 38A1 and 19h9 PD-L1 domains described herein, the bi-epitope CCR comprises two CCR comprising SP- (38 A1 scFv) - (CD 28 hinge and transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (19 h9 scFv) - (CD 28 hinge and transmembrane) - (IL-18 RAP intracellular), wherein SP refers to a signal peptide. In one embodiment, the bi-epitope CCR consists of a sequence comprising SEQ ID NO: 622. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a nucleotide sequence encoding a region of at least 99% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has at least 98% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a region of at least 97% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has at least 96% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a nucleotide sequence encoding a region of at least 95% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a nucleotide sequence encoding a region of at least 94% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a nucleotide sequence encoding a region of at least 93% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a region of at least 92% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a nucleotide sequence encoding a region of at least 91% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has at least 90% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has a nucleotide sequence encoding a region of at least 85% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:622 has at least 80% identity. The foregoing embodiments may include or omit fluorescent proteins, such as eGFP, as needed for analytical purposes.

In one embodiment, the bi-epitope CCR comprises two CCR's comprising SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2rβ transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-2rγ transmembrane and intracellular), SP referring to the signal peptide. In one embodiment, the bi-epitope CCR consists of a sequence comprising SEQ ID NO: 622. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 99% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 98% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 97% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 96% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 95% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has a region of at least 94% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 93% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 92% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 91% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 90% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 has at least 85% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:623 having a region of at least 80% identity. The foregoing embodiments may include or omit fluorescent proteins, such as eGFP, as needed for analytical purposes.

In one embodiment, the bi-epitope CCR comprises two CCRs comprising SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18R 1-transmembrane and intracellular) -T2A-SP- (anti-TROP-2 scFv) - (CD 8 hinge) - (IL-18 RAP-transmembrane and intracellular), wherein SP refers to the signal peptide. In one embodiment, the bi-epitope CCR consists of a sequence comprising SEQ ID NO:624, a nucleotide sequence encoding a polypeptide. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 99% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 98% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a region of at least 97% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 96% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 95% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 94% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 93% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 92% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 91% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a region of at least 90% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 85% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:624 has a nucleotide sequence encoding a region of at least 80% identity. The foregoing embodiments may include or omit fluorescent proteins, such as eGFP, as needed for analytical purposes.

In one embodiment, the bi-epitope CCR comprises two CCR's comprising SP- (cad 47a6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-2rβ intracellular) -T2A-SP- (KM 4097 scFv) - (CD 28 hinge and transmembrane) - (IL-2rγ intracellular), SP referring to the signal peptide. In one embodiment, the bi-epitope CCR consists of a sequence comprising SEQ ID NO:625, and a nucleotide sequence encoding the same. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 99% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 98% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 97% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 96% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 95% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 94% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 93% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 92% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 91% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 90% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 85% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:625 have a region of at least 80% identity. The foregoing embodiments may include or omit fluorescent proteins, such as eGFP, as needed for analytical purposes.

In one embodiment, the bi-epitope CCR comprises two CCR's comprising SP- (cad 47a6.4 scFv) - (CD 28 hinge-transmembrane) - (IL-18R 1 intracellular) -T2A-SP- (KM 4097 scFv) - (CD 28 hinge-transmembrane) - (IL-18 RAP intracellular), wherein SP refers to the signal peptide. In one embodiment, the bi-epitope CCR consists of a sequence comprising SEQ ID NO: 626. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 99% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 98% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 97% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 96% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 95% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 94% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626 having a region of at least 93% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 92% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626 having a region of at least 91% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 90% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region having at least 85% identity. In one embodiment, the bi-epitope CCR consists of a sequence comprising a sequence identical to SEQ ID NO:626, a region of at least 80% identity. The foregoing embodiments may include or omit fluorescent proteins, such as eGFP, as needed for analytical purposes.

In one embodiment, the CCR of the present invention comprises the amino acid sequence of SEQ ID NO: 658. SEQ ID NO: 659. SEQ ID NO: 660. SEQ ID NO: 661. SEQ ID NO:662 or SEQ ID NO:663 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO: 658. SEQ ID NO: 659. SEQ ID NO: 660. SEQ ID NO: 661. SEQ ID NO:662 or SEQ ID NO:663, has at least 99% identity to the sequence set forth in SEQ ID NO: 658. SEQ ID NO: 659. SEQ ID NO: 660. SEQ ID NO: 661. SEQ ID NO:662 or SEQ ID NO:663, has at least 98% identity to the sequence set forth in SEQ ID NO:562, having at least 97% identity to the sequence given in SEQ ID NO: 658. SEQ ID NO: 659. SEQ ID NO: 660. SEQ ID NO: 661. SEQ ID NO:662 or SEQ ID NO:663, has at least 96% identity to the sequence set forth in SEQ ID NO: 658. SEQ ID NO: 659. SEQ ID NO: 660. SEQ ID NO: 661. SEQ ID NO:662 or SEQ ID NO:663, has at least 95% identity to the sequence set forth in SEQ ID NO:562, has at least 90% identity to the sequence given in SEQ ID NO: 658. SEQ ID NO: 659. SEQ ID NO: 660. SEQ ID NO: 661. SEQ ID NO:662 or SEQ ID NO:663 has at least 85% identity or NO: 658. SEQ ID NO: 659. SEQ ID NO: 660. SEQ ID NO: 661. SEQ ID NO:662 or SEQ ID NO:663 has an amino acid sequence with at least 80% identity.

In one embodiment, more than one CCR is encoded by multiple transgenes in a polycistronic vector. In one embodiment, at least one chemokine receptor and at least one CCR are encoded by multiple transgenes in a polycistronic vector. In one embodiment, at least two chemokine receptors and at least one CCR are encoded by multiple transgenes in a polycistronic vector. In one embodiment, at least one chemokine receptor and at least two CCR are encoded by multiple transgenes in a polycistronic vector. In any of the preceding embodiments, the CCR and/or chemokine receptor is encoded by a bicistronic vector. Suitable polycistronic vectors are described herein and in Liu et al, scientific Reports 2017,7 (1), 2193; kim et al, PLoS One 2011,6 (4), e18556, the disclosure of which is incorporated herein by reference in its entirety. Embodiments herein may also employ IRES technology to achieve polycistronic vector designs.

In one embodiment, the CCR of the present invention is a bi-epitope CCR comprising 2 CCR binding to different epitopes of the same target. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein each CCR binds to a different epitope of the target. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises a first scFv domain that binds to a first epitope of a target selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, FR alpha, EPCAM (CD 326), PD-1, PD-L1 (CD 274), PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, and MUC16, and a second CCR comprises a second scFv domain that binds to a different epitope of the target. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of PD-L1 and a second CCR comprises an scFv domain that binds a second epitope of PD-L1. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of PD-1 and a second CCR comprises an scFv domain that binds a second epitope of PD-1. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of CEA and a second CCR comprises an scFv domain that binds a second epitope of CEA. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of CD73 and a second CCR comprises an scFv domain that binds a second epitope of CD 73. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of TROP-2 and a second CCR comprises an scFv domain that binds a second epitope of TROP-2. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of a tissue factor and a second CCR comprises an scFv domain that binds a second epitope of the tissue factor. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of LFA-1 and a second CCR comprises an scFv domain that binds a second epitope of LFA-1. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of FAP and a second CCR comprises an scFv domain that binds a second epitope of FAP. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of VISTA and a second CCR comprises an scFv domain that binds a second epitope of VISTA. In one embodiment, two CCRs are encoded by a bicistronic vector, wherein a first CCR comprises an scFv domain that binds a first epitope of LRRC15 and a second CCR comprises an scFv domain that binds a second epitope of LRRC 15.

In one embodiment, the invention includes a method of treating cancer by administering to a patient in need of treatment a population of tumor-infiltrating lymphocytes (TIL), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs), wherein the TIL, MILs, or PBLs are genetically modified to express two chimeric co-stimulatory receptors (CCR), each CCR comprising:

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, the cancer being treated by administration of a population of TILs, the method comprising:

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

(d) Genetically modifying the second TIL population to express CCR;

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

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the protein of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to an epitope of a protein of interest selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, fra, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, IL-13 ra 2, FAS, tgfbrii and MUC 16.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the protein of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, the extracellular domain being selected from the group consisting of a PD-1 domain, a FAS domain and a tgfbetarii domain.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of a protein of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to an epitope of a protein of interest selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, fra, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, IL-13 ra 2, FAS, tgfbrii and MUC16, the intracellular domains being selected from the group consisting of: CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, CD40L, STAT3, IL-2Rβ, IL-2Rγ, IL-18R1, IL-18RAP, IL-7Rα, IL-12R1, IL-12R2, IL-15Rα, IL-21R, LTBR, and combinations thereof.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to two different epitopes of PD-L1.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to two different epitopes of TROP-2.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to two different epitopes of PD-L1 on each CCR, the intracellular domains being IL-18R1 and IL-18RAP on each CCR.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to two different epitopes of TROP-2 on each CCR, the intracellular domains being IL-18R1 and IL-18RAP on each CCR.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to two different epitopes of PD-L1 on each CCR, the intracellular domains being IL-2rβ and IL-2 γ on each CCR.

a) An extracellular domain;

b) A hinge domain;

c) A transmembrane domain; and

d) At least one intracellular domain;

wherein each extracellular domain binds to a different epitope of the antigen of interest to form a bi-epitope complex and each intracellular domain is selected to provide a secondary unit for signaling, each extracellular domain comprising an scFv binding domain that binds to two different epitopes of TROP-2 on each CCR, the intracellular domains being IL-2rβ and IL-2 γ on each CCR.

IX. chemokine receptors

In some embodiments, the foregoing manufacturing processes for manufacturing TILs, MILs, and PBLs include generation 2 and generation 3 and other processes that may be modified to include steps that include viral or non-viral transduction of the TIL, MILs, or PBLs to express more than one chemokine receptor (also referred to as a chemoattractant cytokine receptor). Chemokine receptors typically have a rhodopsin-like 7-transmembrane (7 TM) structure, interact with chemokines, transduce intracellular signals by coupling with G-proteins and mediate chemotaxis, as described by Murdoch and Finn, blood 2000,95,3032-3043, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the TIL, MILs, or PBL is modified to express a chemokine receptor. In one embodiment, the TIL, MILs, or PBLs are modified to express chemokine receptors and CCR. In one embodiment, the TIL, MILs, or PBLs are modified to express chemokine receptors and CCR, the modification occurring separately. In one embodiment, the TIL, MILs, or PBLs are modified to express chemokine receptors and CCR, the modification occurring simultaneously. In one embodiment, the TIL, MILs, or PBLs are modified to express chemokine receptors but are not also modified to express CCR. TIL, MILs, and PBL can be genetically modified with or separately from the CCR described herein using the chemokine receptors described herein.

A. Chemokine receptor domains

In one embodiment, the TIL, MIL or PBL is transiently or stably modified to express the C-X-C (or CXC) moduleSomatic chemokine receptors, such as CXCR1, CXCR2, CXCR3, CXCR4, or CXCR5. In one embodiment, the TIL, MIL or PBL is transiently or stably modified to express the C-C motif chemokine receptor. Suitable C-C motif chemokine receptors are CCR2, CCR4, CCR6, CCR7 and CCR8. The designation "CCR" such as CCR2, CCR4, CCR6, CCR7 and CCR8 used in combination with the C-C motif chemokine receptor should not be confused with the abbreviation CCR for co-stimulatory chimeric receptors used herein. For example, the term "CCR2" refers herein to the C-C motif chemokine receptor 2, the term "CCR4" refers herein to the C-C motif chemokine receptor 4, the term "CCR6" refers herein to the C-C motif chemokine receptor 6, the term "CCR7" refers herein to the C-C (or CC) motif chemokine receptor 7, and the term "CCR8" refers herein to the C-C motif chemokine receptor 8. In some embodiments, the TIL, MIL, or PBL population is genetically modified to express full length chemokine receptors to induce a chemotactic response and Ca on the TIL, MIL, or PBL population when subjected to ligand gradients ²⁺ Flux to improve tumor tissue movement. In some embodiments, the TIL, MIL, or PBL population is genetically modified to express a C-X-3-C (or CX 3C) motif chemokine receptor. In some embodiments, the TIL, MIL, or PBL population is genetically modified to express an X-C (or XC) motif chemokine receptor. The role of chemokine receptors in T cell homing is described in Sackstein et al, lab. Invest.2017,97,669-97, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the chemokine receptor is a C-X-C motif chemokine receptor. In some embodiments, the chemokine receptor is a C-C motif chemokine receptor. In some embodiments, the chemokine receptor is a C-X-3-C motif chemokine receptor. In some embodiments, the chemokine receptor is an X-C motif chemokine receptor. In some embodiments, the chemokine receptor is selected from CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR6, CCR7, CCR8, and combinations thereof. In some embodiments, the chemokine receptor is selected from CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof.

In some embodiments, the chemokine receptor is selected from the chemokine receptors set forth in table 64.

Table 64: amino acid sequences of exemplary chemokine receptor domains

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In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:627 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:627, having at least 99% identity to the sequence set forth in SEQ ID NO:627, having at least 98% identity to the sequence set forth in SEQ ID NO:627, having at least 97% identity to the sequence set forth in SEQ ID NO:627, having at least 96% identity to the sequence set forth in SEQ ID NO:627, has at least 95% identity to the sequence set forth in SEQ ID NO:627, having at least 90% identity to the sequence set forth in SEQ ID NO:627, or a sequence having at least 85% identity to SEQ ID NO:627, the sequence having an amino acid sequence with at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:627 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:628 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes to SEQ ID NO:628, has at least 99% identity to the sequence set forth in SEQ ID NO:628, has at least 98% identity to the sequence set forth in SEQ ID NO:628, has at least 97% identity to the sequence set forth in SEQ ID NO:628, has at least 96% identity to the sequence set forth in SEQ ID NO:628, has at least 95% identity to the sequence set forth in SEQ ID NO:628, has at least 90% identity to the sequence set forth in SEQ ID NO:628, or a sequence having at least 85% identity to SEQ ID NO:628 has an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:628 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:629 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:629, having at least 99% identity to the sequence set forth in SEQ ID NO:629, having at least 98% identity to the sequence set forth in SEQ ID NO:629, having at least 97% identity to the sequence set forth in SEQ ID NO:629, having at least 96% identity to the sequence set forth in SEQ ID NO:629, having at least 95% identity to the sequence set forth in SEQ ID NO:629, having at least 90% identity to the sequence set forth in SEQ ID NO:629, or a sequence having at least 85% identity to SEQ ID NO:629 has an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:629 or a conservative amino acid substitution or fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:630 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:630, having at least 99% identity to the sequence set forth in SEQ ID NO:630, having at least 98% identity to the sequence set forth in SEQ ID NO:630, has at least 97% identity to the sequence set forth in SEQ ID NO:630, having at least 96% identity to the sequence set forth in SEQ ID NO:630, has at least 95% identity to the sequence set forth in SEQ ID NO:630, has at least 90% identity to the sequence set forth in SEQ ID NO:630, or a sequence having at least 85% identity to SEQ ID NO:630, an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:630 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:631 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes to SEQ id no:631, having at least 99% identity to the sequence set forth in SEQ ID NO:631, having at least 98% identity to a sequence set forth in SEQ ID NO:631, having at least 97% identity to SEQ ID NO:631, having at least 96% identity to the sequence set forth in SEQ ID NO:631, having at least 95% identity to the sequence set forth in SEQ ID NO:631, having at least 90% identity to the sequence set forth in SEQ ID NO:631, or a sequence having at least 85% identity to SEQ ID NO:631, and an amino acid sequence having at least 80% identity thereto. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:631 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:632 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:632, has at least 99% identity to the sequence set forth in SEQ ID NO:632, has at least 98% identity to the sequence set forth in SEQ ID NO:632, has at least 97% identity to the sequence set forth in SEQ ID NO:632, having at least 96% identity to the sequence set forth in SEQ ID NO:632, has at least 95% identity to the sequence set forth in SEQ ID NO:632, has at least 90% identity to the sequence set forth in SEQ ID NO:632, or a sequence having at least 85% identity to SEQ ID NO:632 has an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:632 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:633 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:633 having at least 99% identity to a sequence set forth in SEQ ID NO:633 having at least 98% identity to a sequence set forth in SEQ ID NO:633, having at least 97% identity to the sequence set forth in SEQ ID NO:633 having at least 96% identity to a sequence set forth in SEQ ID NO:633 having at least 95% identity to the sequence set forth in SEQ ID NO:633, having at least 90% identity to a sequence set forth in SEQ ID NO:633, or a sequence having at least 85% identity to SEQ ID NO:633 having an amino acid sequence with at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:633 or a conservative amino acid substitution or fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:634 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:634, has at least 99% identity to the sequence given in SEQ ID NO:634, has at least 98% identity to the sequence given in SEQ ID NO:634, has at least 97% identity to the sequence given in SEQ ID NO:634, has at least 96% identity to the sequence given in SEQ ID NO:634, has at least 95% identity to the sequence given in SEQ ID NO:634, has at least 90% identity to the sequence given in SEQ ID NO:634 has at least 85% identity to the sequence set forth in SEQ ID NO:634 has an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:634 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:635 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof, or an amino acid sequence that hybridizes with SEQ ID NO:635 has at least 99% identity to the sequence set forth in SEQ ID NO:635 has at least 98% identity to the sequence set forth in SEQ ID NO:635 has at least 97% identity to the sequence set forth in SEQ ID NO:635 has at least 96% identity to the sequence set forth in SEQ ID NO:635 has at least 95% identity to the sequence set forth in SEQ ID NO:635 has at least 90% identity to the sequence set forth in SEQ ID NO:635, or a sequence having at least 85% identity to SEQ ID NO:635 has an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:635 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:636 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:636 has at least 99% identity to the sequence set forth in SEQ ID NO:636 has at least 98% identity to the sequence set forth in SEQ ID NO:636 has at least 97% identity to the sequence set forth in SEQ ID NO:636 has at least 96% identity to the sequence set forth in SEQ ID NO:636 has at least 95% identity to the sequence set forth in SEQ ID NO:636 has at least 90% identity to the sequence set forth in SEQ ID NO:636, or a sequence having at least 85% identity to SEQ ID NO:636 has an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:636 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:637 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:637, having at least 99% identity to the sequence set forth in SEQ ID NO:637 has at least 98% identity to the sequence given in SEQ ID NO:637, having at least 97% identity to the sequence set forth in SEQ ID NO:637, having at least 96% identity to the sequence set forth in SEQ ID NO:637 has at least 95% identity to the sequence given in SEQ ID NO:637 has at least 90% identity to the sequence given in SEQ ID NO:637 has at least 85% identity to the sequence set forth in SEQ ID NO:637 to seq id No. s. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:637 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:638 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:638, has at least 99% identity to the sequence set forth in SEQ ID NO:638, has at least 98% identity to the sequence set forth in SEQ ID NO:638, has at least 97% identity to SEQ ID NO:638, has at least 96% identity to the sequence set forth in SEQ ID NO:638, has at least 95% identity to the sequence set forth in SEQ ID NO:638, has at least 90% identity to the sequence set forth in SEQ ID NO:638, or a sequence having at least 85% identity to SEQ ID NO:638 have an amino acid sequence with at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:638 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:639 or a conservative amino acid substitution or fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:639 has at least 99% identity to the sequence given in SEQ ID NO:639 has at least 98% identity to the sequence given in SEQ ID NO:639 has at least 97% identity to the sequence given in SEQ ID NO:639 has at least 96% identity to the sequence given in SEQ ID NO:639 has at least 95% identity to the sequence given in SEQ ID NO:639 has at least 90% identity to the sequence given in SEQ ID NO:639 has at least 85% identity to the sequence set forth in SEQ ID NO:639 to seq id No. s. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:639 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:640 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:640, has at least 99% identity to the sequence set forth in SEQ ID NO:640, has at least 98% identity to the sequence set forth in SEQ ID NO:640, has at least 97% identity to the sequence set forth in SEQ ID NO:640, has at least 96% identity to the sequence set forth in SEQ ID NO:640, has at least 95% identity to the sequence set forth in SEQ ID NO:640, has at least 90% identity to the sequence set forth in SEQ ID NO:640, or a sequence having at least 85% identity to SEQ ID NO:640 has an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:640 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:641 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or a sequence identical to SEQ id no:641 has at least 99% identity to the sequence set forth in SEQ ID NO:641 has at least 98% identity to the sequence set forth in SEQ ID NO:641 has at least 97% identity to the sequence set forth in SEQ ID NO:641 has at least 96% identity to the sequence set forth in SEQ ID NO:641 has at least 95% identity to the sequence set forth in SEQ ID NO:641 has at least 90% identity to the sequence set forth in SEQ ID NO:641 has at least 85% identity to the sequence set forth in SEQ ID NO:641 has an amino acid sequence that is at least 80% identical. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:641 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:642 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence that hybridizes with SEQ ID NO:642, having at least 99% identity to SEQ ID NO:642, having at least 98% identity to SEQ ID NO:642, having at least 97% identity to SEQ ID NO:642, having at least 96% identity to SEQ ID NO:642, having at least 95% identity to SEQ ID NO:642, having at least 90% identity to SEQ ID NO:642, or a sequence having at least 85% identity to SEQ ID NO:642 has an amino acid sequence with at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:642 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:643 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof, or an amino acid sequence identical to SEQ ID NO:643, having at least 99% identity to SEQ ID NO:643, having at least 98% identity to SEQ ID NO:643, having at least 97% identity to SEQ ID NO:643, having at least 96% identity to SEQ ID NO:643, having at least 95% identity to SEQ ID NO:643, having at least 90% identity to the sequence set forth in SEQ ID NO:643, or a sequence having at least 85% identity to SEQ ID NO:643 has an amino acid sequence with at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:643 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:644 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:644 has at least 99% identity to the sequence given in SEQ ID NO:644 has at least 98% identity to the sequence given in SEQ ID NO:644 has at least 97% identity to the sequence given in SEQ ID NO:644 has at least 96% identity to the sequence given in SEQ ID NO:644 has at least 95% identity to the sequence given in SEQ ID NO:644 has at least 90% identity to the sequence given in SEQ ID NO:644 has at least 85% identity to the sequence given in SEQ ID NO:644 have an amino acid sequence having at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:644 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof.

In one embodiment, a chemokine receptor of the invention comprises a domain comprising the sequence of SEQ ID NO:645 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or an amino acid sequence identical to SEQ ID NO:645 has at least 99% identity to the sequence set forth in SEQ ID NO:645 has at least 98% identity to the sequence set forth in SEQ ID NO:645 has at least 97% identity to the sequence set forth in SEQ ID NO:645 has at least 96% identity to the sequence set forth in SEQ ID NO:645 has at least 95% identity to the sequence set forth in SEQ ID NO:645 has at least 90% identity to the sequence set forth in SEQ ID NO:645, or a sequence having at least 85% identity to SEQ ID NO:645 has an amino acid sequence of at least 80% identity. In one embodiment, the chemokine receptor of the invention comprises a nucleotide domain encoding the sequence of SEQ ID NO:645 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof.

In some embodiments, the chemokine receptor is a protein encoded by a nucleotide encoding a C-X-C motif chemokine receptor. In some embodiments, the chemokine receptor is a protein encoded by a nucleotide encoding a C-C motif chemokine receptor. In some embodiments, the chemokine receptor is a protein encoded by a nucleotide encoding a C-X-3-C motif chemokine receptor. In some embodiments, the chemokine receptor is a protein encoded by a nucleotide encoding an X-C motif chemokine receptor. In some embodiments, the chemokine receptor is selected from CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR6, CCR7, CCR8, and combinations thereof. In some embodiments, the chemokine receptor is selected from CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof.

In some embodiments, the chemokine receptor is a protein encoded by a nucleotide set forth in table 65.

Table 65: exemplary nucleotide sequences encoding exemplary chemokine receptor domains

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In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:646 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 99% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 98% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 96% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 95% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 90% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 85% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:646 has at least 80% identity to the amino acid domain encoded by the nucleotide sequence. In embodiments including the preceding embodiments, SEQ ID NO:646 to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:647 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:647 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:647 is codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:648 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 99% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 96% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 95% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 85% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:648 has at least 80% identity to the amino acid domain encoded by the nucleotide sequence. In embodiments including the preceding embodiments, SEQ ID NO:648 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:649 or a conservative amino acid substitution or fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 99% identity to the amino acid domain encoded by a nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 98% identity to the amino acid domain encoded by a nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 97% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 96% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 95% identity to the amino acid domain encoded by a nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 90% identity to the amino acid domain encoded by a nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 85% identity to the nucleotide sequence encoding the amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:649 has at least 80% identity to the amino acid domain encoded by a nucleotide sequence. In embodiments including the preceding embodiments, SEQ ID NO:649 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:650 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 95% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:650 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:650 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:651 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 have at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 have at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 has at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 have at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 have at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 have at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 has at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:651 have at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:651 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:652 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has an amino acid domain encoded by a nucleotide sequence having at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has an amino acid domain encoded by a nucleotide sequence having at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has an amino acid domain encoded by a nucleotide sequence having at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has at least 96% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has at least 95% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has an amino acid domain encoded by a nucleotide sequence having at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has an amino acid domain encoded by a nucleotide sequence having at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:652 has at least 80% identity to the amino acid domain encoded by the nucleotide sequence. In embodiments including the preceding embodiments, SEQ ID NO:652 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:653 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 99% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 98% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 97% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 96% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 95% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 90% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 85% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:653 have an amino acid domain encoded by a nucleotide sequence that is at least 80% identical. In embodiments including the preceding embodiments, SEQ ID NO:653 are codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:654 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:654 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:654 codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:655 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:655 to a nucleotide sequence having at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:655 are codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:656 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has an amino acid domain encoded by a nucleotide sequence of at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has an amino acid domain encoded by a nucleotide sequence of at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has an amino acid domain encoded by a nucleotide sequence having at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has an amino acid domain encoded by a nucleotide sequence of at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has an amino acid domain encoded by a nucleotide sequence of at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has an amino acid domain encoded by a nucleotide sequence having at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has an amino acid domain encoded by a nucleotide sequence of at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:656 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:656 are codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:657 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 99% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 96% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 95% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 90% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 85% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:657 have at least 80% identity to the amino acid domain encoded by the nucleotide sequence. In embodiments including the preceding embodiments, SEQ ID NO:657 codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:658 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:658 has an amino acid domain encoded by a nucleotide sequence having at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:658 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:659 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 99% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 98% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 97% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 96% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 95% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 90% identity to the nucleotide sequence encoding the amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 85% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:659 have at least 80% identity to the nucleotide sequence encoding the amino acid domain. In embodiments including the preceding embodiments, SEQ ID NO:659 codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:660 or a conservative amino acid substitution or fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 99% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 96% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 95% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 90% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 85% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:660 has at least 80% identity to the amino acid domain encoded by the nucleotide sequence. In embodiments including the preceding embodiments, SEQ ID NO:660 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:661 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:661 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:661 is codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:662 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:662 have at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:662 are codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:663 or a conservative amino acid substitution or fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 99% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 98% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 97% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 96% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 95% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 90% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 85% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:663 has at least 80% identity to the nucleotide sequence encoded amino acid domain. In embodiments including the preceding embodiments, SEQ ID NO:663 are codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:664 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has an amino acid domain encoded by a nucleotide sequence that is at least 99% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has an amino acid domain encoded by a nucleotide sequence that is at least 98% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has an amino acid domain encoded by a nucleotide sequence that is at least 97% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has an amino acid domain encoded by a nucleotide sequence that is at least 96% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has an amino acid domain encoded by a nucleotide sequence that is at least 95% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has an amino acid domain encoded by a nucleotide sequence that is at least 90% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has an amino acid domain encoded by a nucleotide sequence that is at least 85% identical. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:664 has at least 80% identity to the amino acid domain encoded by the nucleotide sequence. In embodiments including the preceding embodiments, SEQ ID NO:664 was codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:665 or a conservative amino acid substitution or fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:665 a nucleotide sequence having at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:665 codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:666 or a conservative amino acid substitution thereof or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 97% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 96% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 85% identity to the amino acid domain encoded by the nucleotide sequence. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:666 have at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:666 are codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:667 or a conservative amino acid substitution or fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 have at least 99% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 has at least 98% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 has at least 97% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 have at least 96% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 has at least 95% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 have at least 90% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 has at least 85% identity to the nucleotide sequence encoded amino acid domain. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:667 has at least 80% identity to the nucleotide sequence encoded amino acid domain. In embodiments including the preceding embodiments, SEQ ID NO:667 are codon optimized to improve protein expression.

In one embodiment, the chemokine receptor of the invention comprises an amino acid domain consisting of SEQ ID NO:668 or a conservative amino acid substitution or a fragment, variant or derivative thereof. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 99% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 98% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 97% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 96% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 95% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 90% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 85% identity. In one embodiment, the chemokine receptor of the invention comprises a peptide consisting of a sequence identical to SEQ ID NO:668 has at least 80% identity. In embodiments including the preceding embodiments, SEQ ID NO:668 is codon optimized to improve protein expression.

B. Gene expression method for chemokine receptor

Gene expression methods described elsewhere herein or in the art (including but not limited to lentiviruses, retroviruses, and transposon-based systems) can be used to provide stable expression of chemokine receptors in TILs, MILs, or PBLs, such as the exemplary systems described in section VIII.D above, and include promoters, self-cleaving peptides, linkers, regulatory elements or domains, and other vector components or domains.

The nucleotide sequences of the vectors encoding the exemplary CCR of the present invention are provided in table 66. In one embodiment, the nucleotide sequence of table 66 is codon optimized to improve protein expression. In one embodiment, the nucleotide sequence of table 66 is further modified to include an alternative promoter or regulatory domain as described elsewhere herein. In one embodiment, the nucleotide sequence of table 66 is used in a retroviral expression system. In one embodiment, the nucleotide sequence of table 66 is used in a retroviral expression system using an additional plasmid. Additional details are described in Hawley et al, gene Ther.1994,1,136-38; the disclosure of which is incorporated herein by reference in its entirety.

Exemplary carrier designs for the carriers provided in table 66 are provided in fig. 41 and 42. In one embodiment, the chemokine receptor encoded by the vector shown in fig. 41 is used to genetically modify the TIL products of the invention as described herein. In one embodiment, the chemokine receptor encoded by the vector shown in fig. 41 is used to genetically modify the TIL products of the invention as described herein. In one embodiment, the chemokine receptor encoded by the vector shown in fig. 42 is used to genetically modify the TIL products of the invention as described herein.

Table 66: nucleotide sequences of exemplary vectors for expression of chemokine receptors

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In one embodiment, the chemokine receptor consists of a polypeptide comprising SEQ ID NO: 669. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a nucleotide sequence encoding a region of at least 99% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 98% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 97% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a nucleotide sequence encoding a region of at least 96% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 95% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 94% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 93% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 92% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 91% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 90% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 85% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:669 has a region of at least 80% identity.

In one embodiment, the chemokine receptor consists of a polypeptide comprising SEQ ID NO: 670. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 99% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 98% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 97% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 96% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 95% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 94% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 93% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 92% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 91% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 90% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 85% identity. In one embodiment, the chemokine receptor consists of a peptide comprising a sequence identical to SEQ ID NO:670 a region of at least 80% identity.

In one embodiment, more than one chemokine receptor is encoded by multiple transgenes in a polycistronic vector. In one embodiment, at least one chemokine receptor and at least one CCR are encoded by multiple transgenes in a polycistronic vector. In one embodiment, at least two chemokine receptors and at least one CCR are encoded by multiple transgenes in a polycistronic vector. In one embodiment, at least one chemokine receptor and at least two CCR are encoded by multiple transgenes in a polycistronic vector. In any of the preceding embodiments, the CCR and/or chemokine receptor is encoded by a bicistronic vector. Suitable polycistronic vectors are described herein and in Liu et al, scientific Reports 2017,7 (1), 2193, the disclosure of which is incorporated by reference in its entirety. Embodiments herein may also employ IRES technology to achieve polycistronic vector designs.

Examples

The embodiments covered herein are now described with reference to the following examples. These embodiments are provided for the purpose of illustration only and the disclosure contemplated herein should not be construed as limited by these embodiments, but rather as embracing any and all modifications that may become apparent from the teachings provided herein.

Example 1: preparation of Medium for Pre-REP and REP Processes

This example describes the procedure for preparing tissue culture media for a protocol involving culturing tumor-infiltrating lymphocytes (TILs) derived from various solid tumors. This medium can be used to prepare any of the TILs described in this application and other examples.

CM1 preparation. The following reagents were removed from refrigeration and warmed 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 the appropriate 0.2 μm filter unit volume to be filtered according to table 67 below. Stored at 4 ℃.

Table 67: preparation of CM1

Composition of the components	Final concentration	Final volume 500mL	Final volume 1L
				RPMI1640	NA	450mL	900mL
Human AB serum, heat inactivated 10%	50mL	100mL
				200mM L-glutamine	2mM	5mL	10mL
55mM BME	55μM	0.5mL	1mL
				50mg/mL gentamicin sulfate	50μg/mL	0.5mL	1mL

On the day of use, the required amount of CM1 was preheated in a 37℃water bath and 6000IU/mL IL-2 was added.

Additional supplements may be made as needed according to table 68.

Table 68: optional additional supplementation of CM1

CM2 preparation. The prepared CM1 was removed from the refrigerator or fresh CM1 was prepared as described in section 7.3 above. Removal from refrigeratorMixing the prepared CM1 with equal volume +.>Mix in sterile medium bottles to make the desired amount of CM2. 3000IU/mL IL-2 to CM2 medium was added on the day of use. A sufficient amount of CM2 containing 3000IU/mL of IL-2 was prepared on the day of use. CM2 media flasks were labeled and stored at 4 ℃ until needed for tissue culture.

CM3 preparation. CM3 was prepared on the day of use. CM3 and AIM-The medium was the same and 3000IU/mL IL-2 was supplemented on the day of use. The amount of CM3 needed for sufficient experiments was prepared by adding IL-2 stock directly to the bottles or bags of AIM-V. Mixing evenly by mild shaking. Immediately after addition to AIM-V, "3000IU/mL IL-2" was marked on the bottle. If there is excess CM3, it is stored in a labeled bottle at 4℃for a expiration date of 7 days after preparation. The medium supplemented with IL-2 was discarded after 7 days of storage at 4 ℃.

CM4 preparation. CM4 was identical to CM3, but was additionally supplemented with 2mM Glutamax ^TM (final concentration). 10mL of 200mM Glutamax was added per 1L of CM3 ^TM . By directly adding IL-2 stock solution and Glutamax ^TM Stock solution was placed into AIM-V bottles or bags to prepare the amount of CM4 necessary for adequate experiments. Mixing evenly by mild shaking. Immediately after addition to AIM-V, "3000IL/mL IL-2 and G1utaMAX" were marked on the bottle. If there is excess CM4, it is stored in a bottle at 4℃and the medium name and its expiration date are indicated (7 days after preparation). The medium supplemented with IL-2 was lost after storage at 4℃for more than 7 days.

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 additional T cell growth factors) in combination with the TIL process of any of the examples herein.

Using the procedure described herein, TIL can be grown from a tumor in the presence of IL-2 at the beginning of culture in one group of experiments, and IL-2 replaced with a combination of IL-2, IL-15 and IL-21 in the other group. Upon completion prior to REP, cultured amplifications, phenotypes, functions (cd107 a+ and IFN- γ) and TCR vβ reservoirs were assessed. IL-15 and IL-21 are described elsewhere herein and in Santegoets et al, J.Transl.Med.,2013,11,37.

The results show that CD4 is observed under IL-2, IL-15 and IL-21 treatment conditions relative to IL-2 alone ⁺ And CD8 ⁺ TIL amplification enhancement in both cells>20%). TIL obtained from IL-2, IL-15 and IL-21 treatment cultures was biased to enrich CD8 of TCR V.beta.reservoirs relative to IL-2 alone cultures ⁺ The population is dominant. IFN-. Gamma.and CD107a were elevated for IL-2, IL-15 and IL-21 treated TILs compared to IL-2 treated TILs alone.

Example 3: qualification of gamma-irradiated peripheral mononuclear cells in separate batches

This example describes a simplified procedure for qualifying individual batches of gamma irradiated peripheral mononuclear cells (PBMCs, also known as monocytes or MNCs), the exemplary methods described herein being used as allogeneic feeder cells.

Each irradiated MNC feeder cell batch was prepared from a respective donor. The ability of each batch or donor to amplify TIL in REP in the presence of purified anti-CD 3 (clone OKT 3) antibody and interleukin-2 (IL-2) was screened individually. In addition, each batch of feeder cells was tested without the addition of TIL to verify that the gamma radiation dose received was sufficient to render it replication impossible.

MNC feeder cells with gamma irradiation and growth arrest are required for TIL REP. The membrane receptor on feeder cells MNC binds to anti-CD 3 (clone OKT 3) antibodies and crosslinks to TIL in REP flasks to stimulate TIL expansion. The feeder cell batches were prepared from white blood cell separation of whole blood taken from individual donors. The white blood cell separation product was subjected to Ficoll-Hypaque centrifugation, washed, irradiated and cryopreserved under GMP conditions.

Importantly, patients receiving TIL therapy must not infuse viable feeder cells as they can cause Graft Versus Host Disease (GVHD). Feeder cells thus stop growth by receiving cell gamma irradiation, resulting in double-stranded DNA breaks and loss of cell viability of MNC cells upon re-culture.

Feeder cell batches were evaluated on two criteria: (1) Their ability to amplify TIL > 100-fold in co-culture, and (2) their inability to replicate.

Feeder cell batches were tested in mini REP format using two primary REP pre-TIL cell lines grown in upright T25 tissue culture flasks. Feeder cell batches were tested against two different TIL cell lines, each of which had a different capacity to proliferate in response to activation in REP. A batch of irradiated MNC feeder cells that have been shown to meet the above criteria were run together as a control group alongside the test batch.

To ensure that the test batches in all single experiments received equivalent tests, enough stock of the same prep TIL cell line was prepared to test all conditions and all feeder cell batches.

There were a total of six T25 flasks per batch of feeder cells tested: prep TIL cell line #1 (2 flasks); prep TIL cell line #2 (2 flasks); and a feeder control group (2 flasks). The feeder cell batches in flasks containing TIL cell lines #1 and #2 were evaluated for their ability to amplify TIL. The replication failure of the feeder cell batch in the feeder cell control flasks was evaluated.

A. Experimental protocol

Day-2/3, thawing TIL cell lines. CM2 medium was prepared and CM2 was warmed in a 37℃water bath. A40 mL 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 TIL cell line name used. From LN2 Two designated prep TIL cell lines were stored and removed and vials transferred to the tissue culture chamber. The vials were placed in sealed, clip-chain storage bags and thawed in a 37 ℃ water bath until a small amount of ice remained.

Using a sterile pipette, the vial contents were immediately transferred to 20mL CM2 in a 50mL conical tube prepared, labeled. The cells were washed with a complement of 40mL of CM2 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.

Duplicate small aliquots (20 μl) were removed and cell counted using an automated cell counter. The count is recorded. When counted, 50mL conical tubes containing TIL cells were placed in a humidified atmosphere at 37℃with 5% CO ₂ In the incubator, the cover is released to allow gas exchange. Cell concentration was determined and TIL was diluted to 1×10 ⁶ Each cell/mL was in CM2 supplemented with 3000IU/mL IL-2.

Incubate in humidified 37℃incubator in as porous 2 mL/well as possible in 24-well tissue culture plates until day 0 of mini REP as needed. Different TIL cell lines were cultured on separate 24-well tissue culture plates to avoid confusion and possible cross-contamination.

Day 0, initial Mini REP . Sufficient CM2 medium is prepared for the number of feeder cell batches to be tested (e.g., 800mL CM2 medium is prepared for testing 4 feeder cell batches at a time). A portion of the aliquots of CM2 prepared as described above was taken and supplemented with 3000IU/mL IL-2 for culturing cells (e.g., to test 4 feeder cell batches at a time, 500mL of CM2 medium containing 3000IU/mL IL-2 was prepared).

Each TIL cell line was treated separately to prevent cross-contamination, and 24-well plates with TIL cultures were removed from the incubator and transferred to BSC.

Using a sterile pipette or 100 to 1000. Mu.L pipette and pipette tip, about 1mL of medium was removed from each well of the desired TIL to be used and placed into the unused well of a 24-well tissue culture plate.

Using a new sterile pipette or 100 to 1000 μl pipette and pipette tip, the remaining medium was mixed with TIL in the well to resuspend the cells, and then the cell suspension was transferred to a 50mL conical tube labeled TIL lot name and recording volume.

The wells were washed with stock medium and the volumes transferred to the same 50mL conical tube. Cells were centrifuged at 400×cf to collect cell pellet. The culture supernatant was aspirated off and the pellet was resuspended in 2 to 5mL of CM2 medium containing 3000IU/mL IL-2, the volume required to be used being based on the number of wells collected and the pellet size, the volume being sufficient to ensure >1.3×10 ⁶ Concentration of individual cells/mL.

The cell suspension was thoroughly mixed and the volume was recorded using a serum pipette. 200 μl was removed and cell counted using an automated cell counter. When counted, 50mL conical tubes containing TIL cells were placed in humidified, 5% CO ₂ In a 37℃incubator, the lid is released to allow gas exchange. The count is recorded.

The 50mL conical tube containing TIL cells was removed from the incubator and the cells were 1.3X10 ⁶ The individual cells/mL concentration was resuspended in warm CM2 supplemented with 3000IU/mL IL-2. The 50mL conical tube was returned to the incubator and the lid was released.

The second TIL cell line repeats the above steps.

Prior to inoculating TIL into T25 flasks for experiments, TIL was diluted 1 as follows: 10 to 1.3X10 ⁵ Final concentration of individual cells/mL.

Preparation of MACS GMP CD pure (OKT 3) working solution. OKT3 stock solution (1 mg/mL) was taken out from the refrigerator at 4℃and placed in BSC. OKT3 was used in the medium of mini REP at a final concentration of 30 ng/mL.

In each T25 flask of the experiment, 20mL required 600ng OKT3; 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 1:100 dilutions were performed at a working concentration of 10. Mu.g/mL (e.g., to test 4 feeder cell batches at a time, 1600. Mu.L of 1mg/mL OKT3 in 1:100 dilutions were prepared: 16. Mu.L of 1mg/mL OKT3+1.584mL of CM2 medium containing 3000IU/mL IL-2).

Preparation of T25 flask. Prior to the preparation of feeder cells, each flask was labeled and filled with CM2 medium. Placing the culture flask into 37 ℃ to humidify 5% CO ₂ The incubator is kept warm to keep the medium warm, waiting for the addition of the remaining components. Once the feeder cells were prepared, the components were added to CM2 in each flask.

Further information is provided in table 69.

Table 69: solution information

Preparation of feeder cells. A minimum of 78X 10 is required for each batch tested by this protocol ⁶ And (3) feeder cells. Every 1mL vial frozen by SDBB has 100 x 10 when frozen ⁶ And (3) survival cells. Assuming self-liquid N ₂ 50% recovery after storage thawing, at least two 1mL vials of feeder cells per batch thawing was recommended to give an estimated 100X 10 ⁶ Individual surviving cells were used for each REP. Alternatively, if 1.8mL vials are supplied, only one vial provides enough feeder cells.

Approximately 50mL of IL-2 free CM2 was pre-heated for each feeder cell batch to be tested prior to thawing the feeder cells. Designated feeder cell batch vials were removed from LN2 storage, placed in a clamp chain storage bag and placed on ice. The vials in the sealed, clip-chain storage bags were thawed by immersing them in a 37 ℃ water bath. The vials were removed from the pinch chain bags, sprayed or wiped with 70% EtOH and transferred to the BSC.

Using a pipette, the contents of the feeder cell vial were immediately transferred to 30mL warmed CM2 in a 50mL conical tube. 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 of warmed CM2 plus 3000IU/mL IL-2. 200 μl was removed and cell counted using an automated cell counter. The count is recorded.

The cells were grown at 1.3X10 ⁷ Each cell/mL was resuspended in warm CM2 plus 3000IU/mL IL-2. TIL cells were isolated from 1.3X10 ⁶ Dilution of individual cells/mL to 1.3X10 ⁵ Individual cells/mL.

Setting co-cultivation. TIL cells were isolated from 1.3X10 ⁶ Dilution of individual cells/mL to 1.3X10 ⁵ Individual cells/mL. 4.5mL of CM2 medium was added to a 15mL conical tube. TIL cells were removed from the incubator and resuspended using a 10mL serum pipette. From 1.3X10 ⁶ Each cell/mL TIL suspension was removed from 0.5mL of cells and added to 4.5mL of medium in a 15mL conical tube. The TIL stock solution vials were returned to the incubator. Mixing well. The second TIL cell line was repeated.

Single feeder cell batch flasks containing pre-warmed medium were transferred from incubator to BSC. Mixed feeder cells were aspirated and placed several times with 1mL pipette tip, and 1mL (1.3X10 ⁷ Cells) feeder cells were batched into individual flasks. mu.L of OKT3 working stock (10. Mu.g/mL) was added to each flask. Two control flasks were returned to the incubator.

1mL (1.3X10) ⁵ ) Is transferred to a correspondingly labeled T25 flask. The flask was returned to the incubator and incubated upright. The perturbation is not performed until day 5. This procedure was repeated for all feeder cell batches tested.

Day 5, medium exchange. CM2 containing 3000IU/mL IL-2 was prepared. Each flask required 10mL. Using a 10mL pipette, 10mL of warmed CM2 containing 3000IU/mL IL-2 was transferred to each flask. The flask was returned to the incubator and incubated upright until day 7. All feeder cell batches tested were repeated.

Day 7, collect. 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 grown at the bottom of the flask.

Cells were resuspended in the remaining medium and mixed well to break any cell clumping using a 10mL serum pipette. After thoroughly mixing the cell suspension by pipette tip, 200 μl was removed for cell counting. TIL was counted using an appropriate standard operating program with an automated cytometer device. The count was recorded on day 7. This procedure was repeated for all feeder cell batches tested.

Replication failure in feeder control flasks was assessed and fold expansion from day 0 was assessed in flasks containing TIL.

On day 7, the feeder cell control flasks were maintained until day 14. After completion of the 7 th day count of feeder cell control flasks, 15mL of fresh CM2 medium containing 3000IU/mL IL-2 was added to each control flask. The control flasks were returned to the incubator and incubated in an upright position until day 14.

On day 14, the prolongation of the feeder cell control flask did not proliferate. 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 flask without disturbing the cells growing at the bottom of the flask. Cells were resuspended in the remaining medium and mixed well to break any cell clumps using a 5mL serum pipette. The volume of each flask was recorded.

After thoroughly mixing the cell suspension by pipette tip, 200 μl was removed for cell counting. TIL was counted using an appropriate standard operating program with an automated cytometer device and the count was recorded. This procedure was repeated for all feeder cell batches tested.

B. Results and acceptance of Standard protocol

Results. The dose of gamma irradiation is sufficient to prevent feeder cells from replicating. All batches were expected to meet the evaluation criteria, and also showed a decrease in total survival number of feeder cells remaining on day 7 of REP culture compared to day 0. All feeder cell batches were expected to meet the evaluation criteria for 100-fold expansion of TIL growth by day 7 of REP culture. The day 14 count of the feeder control flasks was expected to last for the non-proliferative trend seen on day 7.

Acceptance criteria. Each replicated TIL cell line tested for each batch of feeder cells met the following acceptance criteria. The acceptance is two-sided, as shown in table 70 below.

Table 70: standard receiving embodiments

The radiation dose was evaluated for the inability of MNC feeder cells to replicate when cultured in the presence of 30ng/mL OKT3 antibody and 3000IU/mL IL-2. Replication could not be assessed by total viable cell count (TVC) as determined by automated cell count at day 7 and day 14 of REP.

The acceptance criteria were "no growth" indicating that the total number of surviving cells on day 7 and day 14 was not increased compared to the initial number of surviving cells placed in culture on day 0 of REP.

Feeder cells were evaluated for their ability to support TIL expansion. TIL growth was measured as fold expansion of surviving cells from day 0 of REP to day 7 of REP. At day 7, TIL culture achieved a minimum of 100-fold expansion (i.e., greater than 100-fold of the total number of surviving TIL cells put into culture on day 0 of REP) as assessed by automated cell counting.

Tandem testing of MNC feeder cell batches that do not meet the acceptance criteria (Contingency Testing). If the MNC feeder cell batch does not meet any of the acceptance criteria outlined above, the batch will be retested by taking the following steps to exclude simple experimental errors as a cause.

If the batch has two or more remaining satellite test vials, the batch is retested. If the batch has one or no satellite test vials remaining, the batch fails according to the acceptance criteria listed above.

For a good run, the problematic and control batches must meet the acceptance criteria described above. After meeting these criteria, the batch may be released for use.

Example 4: preparation of IL-2 stock solution

This example describes the process of solubilizing purified, freeze-dried recombinant human interleukin-2 into a stock sample suitable for use in further tissue culture protocols, including all those described in this application and examples, including those involving the use of rhIL-2.

Procedure. A0.2% acetic acid solution (HAc) was prepared. 29mL of sterile water was transferred to a 50mL conical tube. 1mL of 1N acetic acid was added to a 50mL conical tube. The tube was inverted 2 to 3 times to mix well. The HAc solution was sterilized by filtration using a Steriflip filter.

PBS containing 1% HSA was prepared. 4mL of 25% HSA stock solution was added to 96mL PBS in 150mL sterile filter units. The solution was filtered and stored at 4 ℃. For each vial of rhIL-2 prepared, the form was filled out.

Preparation of rhIL-2 stock solution (6X 10) ⁶ IU/mL final concentration). Each batch rh1L-2 is different and requires information provided in manufacturer's verification Certificate (COA), such as: 1) mass per vial of rhIL-2 (mg), 2) specific activity of rhIL-2 (IU/mg), and 3) recommended 0.2% HAc reconstitution volume (mL).

The 1% HSA volume required for a rhIL-2 batch can be calculated using the following formula:

for example, the specific activity of 1mg vials was 25X 10 according to the CellGenix rhIL-2 batch 1020121 COA ⁶ IU/mg. It is recommended to reconstruct rhIL-2 in 2mL of 0.2% HAc.

The rubber bottle stopper of the IL-2 vial is wiped by alcohol cotton. Using a 16G needle attached to a 3mL syringe, 0.2% HAc of the suggested volume was injected into the vial. Care is taken not to pull the stopper out when withdrawing the needle. The vials were inverted 3 times and vortexed until all the powder was dissolved. Carefully remove the stopper and place it on a side alcohol cotton. Calculated volumes of 1% hsa were added to the vials.

Storage of RhIL-2 solution. When stored for a short period of time<72 hours), the vials were stored at 4 ℃. When stored for a long time >72 hours), the vials were aliquoted into smaller volumes and stored in-20 ℃ frozen vials until ready for use. Avoiding freeze/thaw cyclesA ring. Expiration was 6 months after the date of preparation. Rh-IL-2 tags include vendor and catalog numbers, lot numbers, expiration dates, operator initials, aliquot concentrations and volumes.

Example 5: cryopreservation process

This example describes a process for cryopreserving TIL prepared by the procedure described herein using a CryoMed controlled speed freezer model 7454 (Thermo Scientific).

The equipment used was as follows: aluminum box holder (compatible with CS750 freezer bag), 750mL bag freezer storage box, low pressure (22 psi) liquid nitrogen bottle, refrigerator, thermocouple sensor (tape for bag), and CryoStore CS750 freezer bag (OriGen Scientific).

The freezing process provides a cooling rate of 0.5 ℃ from nucleation to-20 ℃ and 1 ℃ per minute to-80 ℃ end temperature. The program parameters were as follows: step 1-waiting at 4 ℃; step 2: 1.0deg.C/min to-4deg.C; step 3: 20.0deg.C/min to-45deg.C; step 4: 10.0deg.C/min to-10. 0deg.C; step 5:0.5 ℃/min (chamber temperature) to-20 ℃; and step 6: 1.0deg.C/min (sample temperature) to-80deg.C.

Example 6: exemplary procedure of generation 2 and 3

This example shows the generation 2 and generation 3 processes. The TIL of the 2 nd and 3 rd generation procedures consisted largely of autologous TIL derived from individual patients that had undergone surgical removal of the tumor and subsequent ex vivo expansion. The initial first expansion step of the 3 rd generation procedure was cell culture in the presence of interleukin-2 (IL-2) and monoclonal antibody OKT3, which targets the T cell co-receptor CD3 on the scaffolds of irradiated Peripheral Blood Mononuclear Cells (PBMCs).

The production of the 2 nd generation TIL product consists of two stages: 1) Pre-rapid amplification (pre-REP) and 2) a rapid amplification protocol (REP). During prep, resected tumors were cut into 50 pieces of 2 to 3mm each and incubated with serum-containing medium (containing 10% hub supplemented 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 a large-scale second REP amplification. REP consists of CM2 at a 5L volume supplemented with 3000IU/mL rhIL-25X 10 medium and 150. Mu.g of monoclonal anti-CD 3 antibody (OKT 3) loaded thereon ⁹ Co-culture of irradiated allogeneic PBMC feeder cells to activate 200X 10 or less ⁶ The individual surviving cells from prep consisted of 5 days. On day 16, the culture volume was reduced by 90% and the cell fraction was increased by 1X 10 or more ⁹ Each surviving lymphocyte/flask was split into multiple G-Rex-500 flasks and complemented with CM4 to 5L. TIL was incubated for an additional 6 days. REP was collected, washed, formulated on day 22 and cryopreserved prior to delivery to the clinical center for infusion at-150 ℃.

The production of the 3 rd generation TIL product consists of three stages: 1) an initial first amplification protocol, 2) a rapid second amplification protocol (also known as rapid amplification period or REP), and 3) a secondary culture nutrient flask. To affect the proliferation of the initial first amplified TIL, resected tumors were cut into 120 fragments of 2 to 3mm each. On day 0 of initial first amplification, about 100cm in each of 3 100MCS containers ² Build up on the surface area of the support of OKT-3 of about 2.5X10 ⁸ Feeder cell layers of irradiated allogeneic PBMC feeder cells. Tumor fragments were distributed and cultured in 3 100MCS containers each having 500mL serum-containing CM1 medium and 6,000IU/mL interleukin-2 (IL-2) and 15ug OKT-3 for a period of 7 days. On day 7, REP was obtained by introducing about 5X 10 carrying OKT-3 ⁸ Additional feeder cell layers of irradiated allogeneic PBMC feeder cells were initiated with 500mL CM2 medium and 6,000IU/mL IL-2 and 30 μg OKT-3 culture to a tumor-breaking culture period in each of the 3 100MCS containers. REP initiation was enhanced by activating the entire initial first expansion culture in the same vessel, transferring OKT 3-loaded feeder cell fluid to the 100MCS vessel using a closed system. For passage 3, the scale-up or spiking of TILs involves the process steps of expanding whole cell cultures by closed system fluid transfer and transferring to a larger vessel (from 100M flask to 500M flask) and adding an additional 4L of CM4 medium. REP cells were collected, washed, formulated on day 16 and cryopreserved prior to delivery to the clinical center for infusion at-150 ℃.

Overall, the 3 rd generation process is a shorter time, more scalable and easily improved amplification platform, as summarized in Table 71.

Table 71: comparison of exemplary 2 nd generation and exemplary 3 rd generation manufacturing processes

On day 0, tumors of both courses were washed 3 times and fragments were randomly split into two pools; each process is a pool. For passage 2, the fragment was transferred to a GREX 100MCS flask with 1L of CM1 medium containing 6,000IU/mL rhIL-2. In the 3 rd generation procedure, the fragment was transferred to a kit with 500mL containing 6,000IU/mL rhIL-2, 15ug OKT-3 and 2.5X10 ⁸ The cells were cultured in CM 1G-Rex 100MCS flasks. The inoculation of TIL for Rep initiation days occurs on different days according to each process. In the 2 nd generation procedure, the G-Rex 100MCS flask was 90% reduced in volume and the collected cell suspension was transferred to a new G-Rex 500MCS on day 11 to add 5X 10 to the flask containing IL-2 (3000 IU/mL) ⁹ REP initiation was initiated in CM2 medium of individual feeder cells and OKT-3 (30 ng/mL). Cells were expanded and plated on day 16 to multiple G-Rex 500MCS flasks with CM4 medium containing IL-2 (3000 IU/mL) as per protocol. Cultures were then harvested and cryopreserved according to protocol on day 22. With the 3 rd generation procedure, REP initiation occurred on day 7, using the same G-Rex 100MCS at REP initiation. Briefly, 500mL containing IL-2 (6000 IU/mL) and 5X 10 ⁸ Individual feeder cells and 30ug of OKT-3 CM2 medium were added to each flask. On days 9 to 11, the culture scale was expanded longitudinally. The whole volume (1L) of G-Rex100M 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 were included in the comparison, two lung tumors (L4054 and L4055) and one melanoma tumor (M1085T).

CM1 (medium 1), CM2 (medium 2) and CM4 (medium 4) media for L4054 and L4055 were prepared in advance and maintained at 4 ℃. Unfiltered CM1 and CM2 media were prepared to compare cell growth with or without filter media.

The medium used for REP initiation and longitudinal expansion of the L4055 tumor was warmed up to 24 hours at 37℃in advance.

Results. In terms of total viable cells achieved, the 3 rd generation results fall within 30% of the 2 nd generation. The 3 rd generation end products exhibited higher IFN-gamma production after restimulation. The 3 rd generation end products exhibited increased clonal diversity as measured by the presence of the total unique CDR3 sequences. The 3 rd generation end products exhibited longer average telomere lengths.

Prep and REP amplification for the 2 nd and 3 rd generation procedures followed the procedure described above. For each tumor, both pools contained an equal number of fragments. Because of the small size of the tumor, the maximum number of fragments per flask is not achieved. The total REP pre-cells (TVCs) were collected and the 2 nd generation process was counted on day 11 and the 3 rd generation process on day 7. To compare the two REP pre-groups, the cell count was divided by the number of fragments provided in culture to calculate the average viable cells per fragment. As shown in table 72 below, the 2 nd generation process consistently grew more cells per fragment than the 3 rd generation process. The number of expected TVCs on day 11 of the 3 rd generation process is calculated by dividing the pre-REP TVC by 7 and multiplying by 11.

Table 72: prep cell count

* L4055 unfiltered Medium

For the 2 nd and 3 rd generation processes, TVCs were counted as process conditions and the percentage of surviving cells per day of the process was generated. At the time of collection, day 22 (passage 2) and day 16 (passage 3) cells were collected and TVC counts were established. The TVC was then divided by the number of fragments provided on day 0 to calculate the average number of viable cells per fragment. The amplification factor was calculated by dividing the collected TVC by the REP-initiated TVC. As presented in table 73, comparing the 2 nd and 3 rd generation processes, the amplification factor of L4054 was similar; in the case of L4055, the amplification factor was higher in the generation 2 process. Specifically, the medium in this case was warmed up to 24 hours before the REP start day. Higher amplification was also observed at M1085T at passage 3. The number of expected TVCs on day 22 of the 3 rd generation process is extrapolated calculated by dividing the REP TVCs by 16 and multiplying by 22.

Table 73: total viable cell count and fold expansion of TIL end product

After collection, the% viability of the final TIL REP product was compared to a release standard and the results are shown in table 74. All conditions for the 2 nd and 3 rd generation procedures exceeded the 70% viability criteria, which was comparable between procedure and tumor.

Table 74: survival of REP (TIL end product)

Since the number of fragments per vial was below the maximum required number, estimated cell counts for each tumor on the collection day were calculated as shown in table 75. The estimation is based on the following expectations: clinical tumors were large enough to be inoculated with 2 or 3 flasks on day 0.

Table 75: extrapolated estimated cell count calculations to fully expand 2 and 3 flasks for the 3 rd generation process

Phenotypic marker comparison of immunophenotyping-TIL end products. Three tumors, L4054, L4055 and M1085T, underwent TIL amplification during passage 2 and passage 3. After collection, the REP TIL end products were subjected to flow cytometry analysis to test purity, differentiation and memory markers. The percentage of TCR a/b+ cells exceeded 90% under all conditions.

TIL collected from the 3 rd generation process showed higher CD8 and CD28 expression compared to TIL collected from the 2 nd generation process. The 2 nd generation process showed a higher percentage of cd4+.

TIL collected from the 3 rd generation process showed higher central memory compartment expression compared to TIL from the 2 nd generation process.

The markers of activation and depletion of TIL from both tumors L4054 and L4055 were analyzed to compare the final TIL products from the generation 2 and generation 3 TIL amplification procedures. The activation and depletion markers between the 2 nd and 3 rd generation processes are comparable.

Interferon gamma secretion upon restimulation. On the collection day (day 22 of generation 2 and day 16 of generation 3), TILs of L4054 and L4055 underwent overnight restimulation of the coated anti-CD 3 plates. Restimulation of M1085T was performed using anti-CD 3, CD28 and CD137 beads. Supernatants were collected after 24 hours of restimulation in all conditions and frozen. The supernatant from both processes was evaluated for ifnγ ELISA analysis at the same time using the same ELISA plate. Among the three analyzed tumors, higher ifnγ production was observed during passage 3.

Measuring IL-2 levels in culture media. To compare IL-2 consumption between the 2 nd and 3 rd generation processes, cell supernatants of tumors L4054 and L4055 at REP initiation, scale up longitudinally, and harvest day were collected. The amount of IL-2 in cell culture supernatants was determined by the method derived from R&Quantitate ELISA kit measurement of D. Overall trend indicated that the 3 rd generation process maintained higher IL-2 concentration compared to the 2 nd generation process. This is probably due to the higher IL-2 concentration at REP initiation (6000 IU/mL) of passage 3 plus the retention effect of the medium throughout the process.

Metabolic substrate and metabolite analysis. The level of a metabolic substrate such as D-glucose and L-glutamine is measured as an alternative indicator of overall medium consumption. Their equivalent metabolites such as lactate and ammonia were measured. Glucose is a monosaccharide in the medium that is utilized by the granosome to produce ATP energy. When glucose is oxidized, lactic acid (lactate is an ester of lactic acid) is produced. Lactate is highly produced during the exponential growth phase of cells. High amounts of lactate have a negative impact on the cell culture process.

On the REP initiation, scale up and collection days, medium used by L4054 and L4055 during passage 2 and 3 was collected. The used media collection days were: day 11, day 16, and day 22 of generation 2; day 3, 7, 11 and 16. The supernatants were analyzed on a CEDEX bioanalyzer for glucose, lactate, glutamine, glutaMax, and ammonia concentrations.

L-glutamine is an essential amino acid that is required for cell culture medium formulations to be unstable. 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 culture medium of the 2 nd and 3 rd generation processes is supplemented with GlutaMax, which is relatively stable in aqueous solution and does not spontaneously degrade. In two tumor cell lines, group 3 showed a decrease in L-glutamine and GlutaMax and an increase in ammonia throughout REP during the process. In group 2, slightly increased ammonia production was observed for constant concentrations of L-glutamine and GlutaMax. The ammonia from the 2 nd and 3 rd generation processes showed a slight difference in L-glutamine degradation as compared to the day of collection.

Telomere repeat of Flow-FISH . The Flow-FISH technique was used to measure the average telomere repeat length over L4054 and L4055 at passage 2 and 3. Determination of Relative Telomere Length (RTL) was calculated using the telomere PNA kit/FITC from DAKO flow cytometry analysis. Generation 3 shows telomere length comparable to generation 2.

CD3 analysis. To determine the clonal diversity of the cellular products produced by each process, the TIL end products collected by L4054 and L4055 were sampled and the clonal diversity analysis was determined by sequencing the CDR3 portion of the T cell receptor.

Table 76 shows a comparison of the percentage of unique CDR3 sequences shared between the 2 nd and 3 rd generation L4054TIL harvest cell products. 199 sequences were shared between the 3 rd and 2 nd generation end products, corresponding to 97.07% of the first 80% from the unique CDR3 sequences shared by the 2 nd and 3 rd generation end products.

Table 76: comparison of shared uCDR3 sequences between L4054 generation 2 and generation 3 Processes

Table 77 shows a comparison of the percentage of unique CDR3 sequences shared between the 2 nd and 3 rd generation L4055TIL harvest cell products. 1833 sequences were shared between the 3 rd and 2 nd generation end products, corresponding to 99.45% of the first 80% from the unique CDR3 sequences shared by the 2 nd and 3 rd generation end products.

Table 77: comparison of shared uCDR3 sequences between L4055 generation 2 and generation 3 Processes

Unfiltered CM1 and CM2 media were prepared in advance and kept at 4 ℃ until used for tumor L4055 for the 2 nd and 3 rd generation processes.

The medium for tumor L4055 was warmed at 37 ℃ for 24 hours before the REP start day of the 2 nd and 3 rd generation processes.

LDH was not measured in the supernatant collected in the process.

M1085T TIL cell counts were performed using a K2 cell counter.

In the case of tumor M1085T, samples such as supernatant for metabolic analysis, TIL products for activation and depletion marker analysis, telomere length and CD3-TCR vb analysis were not available.

Conclusion(s). This example compares the functional quality attributes of 3 independent donor tumor tissues plus the expansion phenotype identification and the medium consumption during passage 2 and 3.

The total surviving cells and total nucleated cell population viability produced were assessed by the pre-REP and REP expansion comparisons of passage 2 and passage 3. TVC cells were not comparable in dose between passage 2 (22 days) and passage 3 (16 days) on the harvest day. The dose of 3 rd generation cells was less than about 40% of the total viable cells collected at the time of collection at 2 nd generation.

Extrapolated cell numbers for the 3 rd generation process were calculated assuming that pre-REP collection occurred on day 11 instead of day 7 and REP collection occurred on day 22 instead of day 16. In both instances, passage 3 shows a closer number of TVCs than passage 2, indicating that early activation may allow for overall better performance of TIL growth.

In the case of extrapolated values of additional flasks (2 or 3) in the 3 rd generation procedure, it is assumed that tumors of larger size are treated and the maximum number of fragments required for the procedure as described is reached. It was observed that TVC at similar doses as compared to day 22 of the generation 2 process could be collected on day 16 of the generation 3 process. This observation is important and indicates that early activation of culture may allow TIL to perform better in less processing time.

The comparison of prep and REP expansion at passage 2 and passage 3 was evaluated with respect to the total surviving and total nucleated cell population produced. TVC cells were not comparable in dose between passage 2 (22 days) and passage 3 (16 days) on the harvest day. The dose of 3 rd generation cells was less than about 40% of the total viable cells collected at the time of collection at 2 nd generation.

For phenotypic identification, higher cd8+ and cd28+ expression was observed for the 3 rd generation process compared to the 2 nd generation process for the three tumors. This data indicates that the 3 rd generation process has improved final TIL product properties compared to the 2 nd generation.

The 3 rd generation process showed slightly higher central memory compartments than the 2 nd generation process.

The generation 2 and generation 3 processes show comparable activation and depletion signatures, although the duration of the generation 3 process is shorter.

Of the three tumors analyzed, ifnγ (ifnγ) of the 3 rd generation end product was produced 3 times higher than the 2 nd generation. This data indicates that the 3 rd generation process produces a TIL product that is highly functional and more efficient than the 2 nd generation process, possibly due to the higher expression of CD8 and CD28 expression of the 3 rd generation. Phenotypic identification suggests that generation 3 has a positive trend in cd8+, cd28+ expression of three tumors compared to generation 2.

The telomere length of the TIL end product between generation 2 and generation 3 is comparable.

The amounts of glucose and lactate between the final products of passage 2 and 3 are comparable, suggesting that the amount of nutrients of the medium of the passage 3 process is not affected, since no volume reduction removal is performed daily in the process and the overall culture area is less in the process compared to passage 2.

The overall 3 rd generation process shows a nearly twice reduction in processing time compared to the 2 nd generation process, which will result in a substantial reduction in the commodity Cost (COG) of TIL products amplified by the 3 rd generation process.

IL-2 consumption indicates an overall trend in IL-2 consumption during passage 2, with IL-2 being higher during passage 3 because old media was not removed.

The 3 rd generation procedure showed higher clonal diversity as measured by CDR3TCRab sequence analysis.

The addition of feeder cells and OKT-3 on day 0 prior to REP allowed the use of early activation TIL and overall preferred TIL growth performance of the 3 rd generation process.

Table 78 describes various embodiments and results of the 3 rd generation process and compares to the current 2 nd generation process.

Table 78: exemplary generation 2 and 3 Process characterization

Example 7: exemplary embodiment of the 3 rd Generation amplification procedure at day 0

Tumor wash media were prepared. The medium was warmed before starting. 5mL of gentamicin (50 mg/mL) was added to a 500mL bottle of HBSS. 5mL of tumor wash medium was added to a 15mL conical tube to be used for OKT3 dilution. Stored at Room Temperature (RT).

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 feeder cells are thawed and then counted.

The concentration of feeder cells is approximately optimally in the range of 5X 10 ⁴ And 5X 10 ⁶ Between individual cells/mL. Four conical tubes containing 4.5mL AIM-V were prepared. Cell fractions of 0.5mL were added to each cell count.

If the total number of surviving feeder cells is not less than 1X 10 ⁹ Individual cells, then proceed to the next step to modulateWhole feeder cell concentration. Calculation for addition of 1×10 ⁹ The individual cells are passed to the second feeder cell bag and the feeder cell volume removed from the first feeder cell bag is required.

Using a p1000 micropipette, 900 μl of tumor wash medium was transferred to OKT3 aliquots (100 μl). Using syringe and sterile technique, 0.6mL of OKT3 was withdrawn and added to the second feeder cell bag. The volume of the medium 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 100uL aliquots at the original stock solution concentration (1 mg/mL) from the vial. About 10X aliquots per 1mL vial. Stored at-80C. Day 0: 15. Mu.g/flask, i.e., 30ng/mL in 500 mL-up to 60. Mu.L of about 1 aliquot.

Tumor samples were prepared. 6-well plates and 100mm dishes (4 total) were obtained. The 6-well plate was labeled "excessive tumor pieces". Each of the four 100mm dishes is labeled "Wash_01", "Wash_02", "Wash_03", "Wash_04", and "Retention".

5mL of tumor wash medium was added to all wells in a six well plate labeled "excess tumor plate". The tumor-maintaining wash medium can be used to further maintain tumor hydration during the segmentation.

50mL of tumor wash medium was added to each 100mm dish labeled "Wash_01", "Wash_02", "Wash_03" and "Retention". Using a marker, each dish was labeled "section 1" through "section 4". The tumors were incubated in "Wash_01" for > 3 min at ambient temperature. The tumors were incubated in "Wash_02" for > 3 min at ambient temperature. The tumors were incubated in "wash_03" for > 3 min at ambient temperature. After washing is complete, the tumor is moved to a "holding" dish to ensure that the tissue remains hydrated.

When tumor incubation was performed, 10mL of tumor transport medium was transferred to a tube labeled "tumor transport medium". 10mL of tumor transport medium was withdrawn to the syringe and 5mL of tumor transport medium was used to inoculate one anaerobic and aerobic sterile flask each.

The whole segmentation process places a ruler below the culture dish cover. Tumor length and number of fragments were measured and recorded. Tumors are divided into four intermediate pieces or groups of four groups of equal volume and the tumor structure of each intermediate piece is preserved. The tumor pieces were kept hydrated.

Any intermediate tumor pieces that are not dividing are transferred to a holding dish to keep the tissue hydrated.

Tumors were segmented into 27mm using the ruler below the segmentation dish lid as a reference ³ Fragments (3X 3 mm). The middle segment is split until 60 segments are reached. The total number of final fragments was counted and G-Rex100MCS flasks were prepared based on the number of final fragments generated (typically 60 fragments per flask).

The preferred tissue fragments are retained in conical tubes labeled "fragment tube 1" through "fragment tube 4". The number of G-Rex100MCS flasks to be inoculated with the feeder cell suspension was calculated based on the number of fragment tubes from which they were derived.

Feeder cell bags were removed from the incubator and inoculated with G-Rex100MCS. Labeled D0 (day 0).

Culture of tumor fragments added to G-Rex-100MCS. Under sterile conditions, the G-Rex100MCS labeled "tumor fragment culture (D0) 1" and the cap of the 50mL conical tube labeled "fragment tube" were loosened. The opened fragment tube 1 was swirled while slightly lifting the lid of the G-Rex100MCS. The medium containing the fragments was added to the G-Rex100MCS at the time of vortexing. The number of fragments transferred to the G-Rex100MCS is recorded.

Once the fragment was at the bottom of the GREX flask, 7mL of medium was withdrawn and seven 1mL aliquots were generated-5 mL for extended identification, and 2mL was used on sterile samples. 5 aliquots (final fragment culture supernatants) for expanded identification were stored at-20℃until needed.

One anaerobic BacT/Alert flask and one aerobic BacT/Alert flask, each containing 1mL of the final fragment culture supernatant, were inoculated. The sampled flasks were repeated.

Example 8: exemplary embodiment of the 3 rd Generation amplification Process on days 7 to 8

Feeder cell bags were prepared. When frozen, the feeder cell bags were thawed in a 37 ℃ water bath for 3 to 5 minutes. Feeder cells were counted if frozen.

The concentration of feeder cells is approximately optimally in the range of 5X 10 ⁴ And 5X 10 ⁶ Between individual cells/mL. Four conical tubes containing 4.5mL AIM-V were prepared. Each cell count was supplemented with 0.5mL of cell fraction to a new frozen vial. Samples were mixed well and cell counts were performed.

If the total number of surviving feeder cells is not less than 2X 10 ⁹ Individual cells, the next step is performed to adjust feeder cell concentration. Calculation for adding 2×10 ⁹ The individual cells are passed to the second feeder cell bag and the feeder cell volume removed from the first feeder cell bag is required.

Using a p1000 micropipette, 900 μl HBSS was transferred to a 100 μl OKT3 aliquot. Mix by pipetting up and down 3 times. Two aliquots were prepared.

OKT3 formulation details: OKT3 may be aliquoted and frozen in 100uL aliquots at the original stock solution concentration (1 mg/mL) from the vial. About 10 x aliquots per 1mL vial. Stored at-80C. Day 7/8: 30. Mu.g/flask, i.e., 60ng/mL in 500 mL-up to 120. Mu.l of about 2 aliquots.

Using syringe and sterile technique, 0.6mL of OKT3 was withdrawn and added to the feeder cell bag, ensuring that all were added. The volume of the medium 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.

Preparation of G-Rex100MCS flask containing feeder cell suspension. The number of G-Rex100MCS flasks to be treated was recorded based on 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.

Removal of supernatant prior to feeder cell suspension addition. A10 mL syringe was connected to the G-Rex100 flask and 5mL of medium was withdrawn. Five 1mL aliquots-5 mL were generated for extensive identification, and 5 aliquots (final fragment culture supernatants) were stored at-20deg.CUntil the test delegate requests for extended authentication. Each G-Rex100 flask was labeled and repeated.

5 to 20 1mL samples were prepared for identification, depending on the number of flasks:

5mL = 1 flask

10mL = 2 flasks

15mL = 3 flasks

20mL = 4 flasks

Feeder cells were continuously seeded into G-Rex100MCS and repeated in each G-Rex100MCS flask. Using a sterile transfer method, 500mL of a second feeder cell bag (assuming 1 g=1 mL) was gravity transferred to each G-Rex100MCS flask by weight and the amount recorded. Cultures indicated as day 7 were repeated in each G-Rex100 flask. The G-Rex100MCS flask was transferred to an incubator.

Example 9: exemplary embodiment of the 3 rd Generation amplification procedure at days 10 to 11

The first G-Rex 100MCS flask was removed and 7mL of the pretreatment culture supernatant was removed using a 10mL syringe using sterile conditions. Seven 1mL aliquots-5 mL were generated for extensive identification and 2mL were used on sterile samples.

The flask was carefully mixed, 10mL of the supernatant removed using a new 10mL syringe and transferred to a 15mL tube labeled "D10/11 mycoplasma supernatant".

Carefully mix the flasks, use a new syringe, remove the following volumes depending on how many flasks are 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 drawn from all flasks and 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 expanded identification were stored at-20℃until needed. One anaerobic BacT/Alert flask and one aerobic BacT/Alert flask, each containing 1mL of pretreatment culture supernatant, were inoculated.

The cell suspension was continuously transferred to G-Rex 500MCS and repeated for each G-Rex 100MCS. Using aseptic conditions, the contents of each G-Rex 100MCS were transferred to G-Rex 500MCS, and about 100mL of fluid transfer was monitored at a time. Transfer was stopped when the volume of the G-Rex 100MCS was reduced to 500 mL.

During the transfer step, 10mL of the cell suspension was drawn from the G-Rex 100MCS using a 10mL syringe to the syringe. The instructions according to the number of flasks in culture were followed. If there are only 1 flask: a total of 20mL was removed using two syringes. If 2 flasks: 10mL was removed per flask. If 3 flasks: 7mL was removed per flask. If 4 flasks: remove 5mL per flask. The cell suspension was transferred to a common 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 was determined as follows:

efficacy assays (such as those described herein) or IFN-gamma or granzyme B assays at a minimum of 10X 10 ⁶ Individual cells

Mycoplasma 1X 10 ⁶ Individual cells

CD3+/CD45+ flow cytometry 5X 10 ⁶ Individual cells

The G-Rex 500MCS flask was transferred to an incubator.

Preparation of QC samples. The assay in this example requires at least 15X 10 ⁸ Individual cells. The included assays were: cell count and viability; mycoplasma (1X 10) ⁶ Individual cells/average viable concentration); streaming (5X 10) ⁶ Individual cells/average viable concentration); IFN-g assay (5X 10) ⁶ Individual cells-1×10 ⁶ A cell; IFN-gamma assays require 8 to 10X 10 ⁶ Individual cells.

At 10×10 ⁶ The volume of the cell fraction used for cryopreservation was calculated per cell/mL and the number of vials to be prepared was calculated.

Example 10: exemplary embodiment of the 3 rd Generation amplification procedure at day 16 to 17

Washing buffer preparation (1%HSA Plasmalyte A). HSA and Plasmalyte were transferred to 5L bags to prepare the LOVO wash buffer. Using aseptic conditions, 25% HSA was transferred to a 5L bag in a total volume of 125 mL. Stored at room temperature.

Remove and transfer 10mL or 40mL of wash buffer to "IL-2 6X 10 ⁴ IU/mL "in tube (10 mL if IL-2 is prepared in advance, or 40mL if IL-2 is freshly prepared).

The volume of reconstituted IL-2 to be added to plasmalyte+1% HSA was calculated: the volume of reconstituted IL-2= (final concentration of IL-2 x final volume)/specific activity of IL-2 (determined based on standards). IL-2 final concentration of 6X 10 ⁴ IU/mL. The final volume was 40mL.

The calculated initial volume of IL-2 required for the reconstituted IL-2 was removed and transferred to "IL-2 6X 10 ⁴ IU/mL "test tube. IL-2 6X 10 from aliquots prepared in advance was added at 100. Mu.L ⁶ Labeling of IU/mL to LOVO wash buffer containing 10mL "IL-2 6X 10 ⁴ IU/mL "test tube.

About 4500mL of supernatant was removed from the G-Rex 500MCS flask. The remaining supernatant was swirled and the cells transferred to a cell collection tank bag. Repeat in all G-Rex 500MCS flasks.

60mL of supernatant was removed and added to supernatant tubes for quality control assays, including mycoplasma detection. Stored at +2 to 8 ℃.

Cell collection. Cells were counted. Four 15mL conical vials containing 4.5mL AIM-V were prepared. These can be prepared in advance. The optimum range is 5×10 ⁴ And 5X 10 ⁶ Between individual cells/mL. (1:10 dilution recommended). 1, the method comprises the following steps: for 10 dilutions, 500 μl of CF was added to 4500 μl of AIM V prepared previously. The dilution factor is recorded.

Calculate TC (total cells) (live+dead) =before LOVO

Average total cells

Concentration (TC concentration before LOVO)

(Living+dead)

X

Volume of Source bag

Calculate TVC (total surviving cells) (viable) =before LOVO

Average total viable cells

Concentration (TVC before LOVO)

(living)

X

Volume of LOVO Source bag

When Total Cell (TC) number>5×10 ⁹ At the time of removal 5X 10 ⁸ Individual cells were cryopreserved as MDA retention samples. 5X 10 ⁸ Mean TC concentration (step 14.44) =volume to be removed.

When the Total Cell (TC) number is less than or equal to 5×10 ⁹ At the time of removal 4X 10 ⁶ Individual cells were cryopreserved as MDA retention samples. 4X 10 ⁶ Average TC concentration = volume to be removed.

The desired volume is removed from the LOVO source bag using a syringe of appropriate size. Remain in the incubator until the cryopreservation step.

When determining the total cell number, the number of cells to be removed should allow for a retention of 150X 10 ⁹ And (3) survival cells. Confirmation of TVC 5 x 10 before LOVO ⁸ Or 4X 10 ⁶ Or not applicable. The volume of cells to be removed is calculated.

The remaining total cells remaining in the bag were calculated. pre-LOVO TC (total cells) was calculated. [ average Total cell concentration x residual volume = residual pre-LOVO TC ]

The procedure corresponding to table 79 was selected based on the total number of cells remaining.

Table 79: total number of cells

Total cells:	retentate (mL)
		0<The total cells are less than or equal to 31 multiplied by 10 ⁹	115
31×10 ⁹ <The total cells are less than or equal to 71 multiplied by 10 ⁹	165
		71×10 ⁹ <The total cells are less than or equal to 110 multiplied by 10 ⁹	215
110×10 ⁹ <The total cells are less than or equal to 115 multiplied by 10 ⁹	265

The volume of IL-2 to be added corresponding to the procedure used is selected. The volume calculation is as follows: retentate volume x 2 x 300IU/mL = IU of desired IL-2. IU/6X10 of IL-2 required ⁴ IU/mL = IL-2 volume to be added to the bag after LOVO. Record all added volumes. Samples obtained in frozen vials were used for further analysis.

The cell products were mixed homogeneously. All bags are sealed for further processing, including cryopreservation as appropriate.

The resulting frozen vial samples were subjected to endotoxin, IFN-gamma, sterility and other optional assays.

Example 11: exemplary 3 rd Generation procedure (also referred to as 3.1 rd Generation)

This example describes a further study on the "comparability between the 2 nd and 3 rd generation TIL amplification procedures". The 3 rd generation process is modified to include an activation step early in the process, which aims at increasing the final Total Viable Cell (TVC) output to be comparable (or better) than the 2 nd generation while maintaining the previously seen phenotype and functional profile.

The scope of this example involves assessing TVC output by introducing an activation step to the cultured tumor fragments on day 0; showing the comparability of the 3 rd generation standard and control group in terms of functional and expanded phenotype identification in two independent patient tumors; and analysis of media consumption and metabolite production to confirm maintenance of the treatment parameters under physiological conditions.

All runs of this example were performed on a full scale platform using commercial donor tumor tissue as starting material.

The 3 rd generation embodiment is modified to a further embodiment, referred to herein in this example as 3.1 rd generation.

In one embodiment, the 3.1 rd generation TIL manufacturing process has four operator interventions:

1) Tumor debris separation and activation: on day 0 of the procedure, tumors were segmented and the resulting final fragments were about 3×3mm each (up to 240 fragments total) and cultured in 1 to 4G-Rex 100MCS flasks. Each flask contained up to 60 fragments, 500mL of CM1 or DM1 medium, and was supplemented with 6,000IU rhIL-2, 15 μg OKT3, and 2.5X10 ⁸ And (c) irradiating the allogeneic mononuclear cells. Cultures were incubated at 37℃for 6 to 8 days.

2) TIL culture reactivation: on days 7 to 8, by slow addition of CM2 or DM1 medium (both supplemented with 6,000IU rhIL-2, 30. Mu.g OKT3 and 5X 10) ⁸ Individual irradiated allogeneic monocytes) to supplement the culture. Care was taken not to disturb the existing cells at the bottom of the flask. Cultures were incubated at 37℃for 3 to 4 days.

3) Cultivation scale is longitudinally enlarged: occurs on days 10 to 11. During the vertical growth of the culture scale, all contents of the G-Rex100MCS were transferred to G-Rex500MCS flasks containing 4L of CM4 or DM2 (both supplemented with 3,000IU/mL of 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 volume of the flask was reduced and combined. Cells were concentrated and washed with PlasmaLyte a pH 7.4 containing 1% HSA. The washed cell suspension was formulated using a CryoStor10 as 1:1 ratio and supplemented with rhIL-2 to a final concentration of 300IU/mL.

DP is a cryopreserved and stored in gas phase liquid nitrogen by controlled rate freezing. * The complete standard TIL Medium 1, 2 or 4 (CM 1, CM2, CM 4) may be substituted with CTS ^TM OpTmizer ^TM T cell noneThe serum amplification medium is referred to as the determination medium (DM 1 or DM 2) as described above.

Description of the procedure. On day 0, tumors were washed 3 times, then breaking into 3× 3X 3 final fragment. Once the whole tumor is broken, the final fragments are then equally randomly grouped and divided into three pools. Each group was imported into a pool of randomly grouped fragments, with the same number of fragments added according to three experimental matrices.

Tumor L4063 amplification was performed using standard media and tumor L4064 amplification was performed using defined media (CTS OpTmizer) throughout the TIL amplification process. The components of the culture medium are described herein.

CM1 complete medium 1: RPMI+Glutamine supplemented with 2mM Glutamax, 10% human AB serum, gentamicin (50 ug/mL), 2-mercaptoethanol (55 uM). 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 (2 mM). The final media formulation was supplemented with 3000IU/mL IL-2.

CTS OpTmizer CTS ^TM OpTmizer ^TM T cell expansion basal medium supplemented with CTS ^TM OpTmizer ^TM T cell expansion supplement (26 mL/L).

DM1：CTS ^TM OpTmizer ^TM T cell expansion basal medium supplemented with CTS ^TM OpTmizer ^TM T cell expansion supplement (26 mL/L) and CTS ^TM Immune cells SR (3%) and containing Glutamax (2 mM). The final formulation was supplemented with 6,000IU/mL IL-2.

DM2：CTS ^TM OpTmizer ^TM T cell expansion basal medium supplemented with CTS ^TM OpTmizer ^TM T cell expansion supplement (26 mL/L) and CTS ^TM Immune cells SR (3%) and containing Glutamax (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 and kept at 4 ℃ in advance until the day before use, warmed up to 24 hours in 37 ℃ incubator in advance before the treatment day.

TIL culture reactivation of both tumors occurred on day 7. The longitudinal expansion of the scale of L4063 occurs on day 10 and L4064 on day 11. Both cultures were collected on day 16 and cryopreserved.

The result achieved. Cell counts and% viability were determined for the 3.0 th and 3.1 th generation processes. Amplification in all conditions followed the details described in this example.

For each tumor, the fragments were divided into an equal number of three pools. Because of the small size of the tumor, the maximum number of fragments per flask is not achieved. The total surviving cells and cell viability of each condition were assessed in three different procedures. Cell counts were judged at day 7 as reactivated TVC, day 10 (L4064) or day 11 (L4063) as scale up longitudinally expanded TVC and day 16/17 as harvested TVC.

Cell counts on days 7 and 10/11 were obtained as FIO. Fold expansion was calculated as the TVC on day 16/17 of collection divided by the TVC on day 7 of 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 viable cells per fragment.

Cell counts and viability assays of L4063 and L4064 were performed. The 3.1 generation-test procedure produced more cells per fragment in both tumors than the 3.0 generation procedure.

Total viable cell count and fold expansion; viability during the procedure%. At reactivation, scale up and collection, all conditions were subjected to percent viability. The% viability of the final TVC was compared to a release standard at day 16/17 of the collection. All conditions evaluated exceeded the 70% viability criteria, comparable between the procedure and the tumor.

Immunophenotyping-phenotypic identification of TIL end products. The final product was subjected to flow cytometry analysis to test purity, differentiation and memory markers. All conditions were consistent with the percentage of TCR α/β, cd4+ and cd8+ cell populations.

Enlarged phenotyping of REP TIL was performed. The TIL products of the 3.1 rd generation condition showed a higher percentage of cd4+ cells than the 3.0 rd generation in both tumors, and the 3.0 rd generation had a higher percentage of cd28+ cells from the cd8+ population than the 3.1 rd generation condition.

TIL collected from the 3.0 th and 3.1 th generation processes showed comparable expression of phenotypic markers such as CD27 and CD56 on cd4+ and cd8+ cells and comparable expression of CD28 on cd4+ gated cell populations. Memory signature comparison of TIL end product:

frozen samples of TIL collected on day 16 were stained for analysis. The state of TIL memory between the 3.0 th and 3.1 th generation processes is comparable. Activation and depletion markers of TIL end products were compared:

the activation and depletion markers are comparable between cd4+ and cd8+ cells that are gated by the 3.0 th and 3.1 th generation processes.

Interferon gamma secretion upon restimulation. The collected TILs of L4063 and L4064 were restimulated overnight using coated anti-CD 3 plates. In both analyzed tumors, higher ifnγ production was observed for the 3.1 generation process compared to the 3.0 generation process.

Measuring IL-2 levels in culture media. To compare the amount of IL-2 consumption between all conditions and processes, cell supernatants were collected and frozen at day 7 initial reactivation, day 10 (L4064)/day 11 (L4063) scale up longitudinally, and day 16/17 collection. The supernatant was subsequently thawed and then analyzed. The amount of IL-2 in the cell culture supernatant was measured by the manufacturer's protocol.

Overall, the 3 rd and 3.1 rd generation processes are comparable in terms of IL-2 consumption during the complete process evaluated between the same medium conditions. IL-2 concentration (pg/mL) analysis of spent media collected by L4063 and L4064.

Metabolite analysis. Spent media supernatants were collected from L4063 and L4064 at the beginning of 7-day reactivation, at day 10 (L4064) or at day 11 (L4063) scale-up and at day 16/17 collection for each of L4063 and L4064. Analysis of the supernatant on a CEDEX bioanalyzerSugar, lactate, glutamine, glutaMax and ammonia concentrations.

The medium was determined to have a higher glucose concentration of 4.5g/L compared to the complete medium (2 g/L). Overall, the concentration and consumption of glucose during the 3.0 th and 3.1 th generation within each media type is comparable.

An increase in lactate was observed in all test conditions for both tumors L4063 and L4064. The increase in lactate was comparable between the 3.0 th and 3.1 st generation conditions and between the two media used for reactivation amplification (complete medium for L4063 and defined medium for L4064).

In the case of L4063, standard basal medium contains 2mM L-glutamine and is supplemented with 2mM GlutaMax to compensate for the natural degradation of L-glutamine to L-glutamic acid and ammonia in culture conditions.

For L4064 tumors, basal medium of defined (serum-free) medium was used which did not contain L-glutamine and was only supplemented with GlutaMax to a final concentration of 2mM. GlutaMax is a dipeptide of L-alanine and L-glutamine that is more stable in aqueous solution than L-glutamine and does not spontaneously degrade to glutamic acid and ammonia. In contrast, the dipeptide gradually dissociates into individual amino acids, thereby maintaining a lower but sufficient concentration of L-glutamine to maintain robust cell growth.

For L4063, the concentrations of glutamine and GlutaMax decreased slightly on the scale up day, but showed an increase to an amount similar or near the reactivation day on the harvest day. With L4064, glutamine and GlutaMax concentrations showed a slight degradation at a similar rate between different conditions during the entire process.

As expected, L4063 (grown in standard medium with 2mM glutamine+2 mM GlutaMax) had a higher ammonia concentration than L4064 (grown in defined medium with 2mM GlutaMax). In addition, ammonia may gradually increase or accumulate during the incubation period, as expected. There was no difference in ammonia concentration between the three different test conditions.

Telomere repeat of Flow-FISH. The Flow-FISH technique was used to measure the average telomere repeat length over L4063 and L4064 at 3 rd and 3.1 rd generation processes. Relative telomere length Determination of (RTL) was calculated using the telomeric PNA kit/FITC from DAKO flow cytometry analysis. Telomere measurement was performed. The telomere lengths of the L4063 and L4064 samples were compared to 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 3 rd and 3.1 rd generation processes evaluated in both tumors showed comparable telomere length. TCR V beta reservoir analysis

To determine the clonal diversity of the cellular products produced by each process, a clonal diversity analysis of the final TIL product was determined by sequencing the CDR3 portion of the T cell receptor.

Three parameters between three conditions were compared:

diversity index of unique CDR3 (uCDR 3)

Shared uCDR3%

In the first 80% of uCDR 3:

comparative shared uCDR3 copy%

Omicron comparison of unique clone type frequencies

Percentage of unique CDR3 sequences shared between L4063 control and TIL cell products collected from 3.1 generation test: 975 sequences were shared between the 3 rd and 3.1 rd generation test end products, corresponding to 88% of the first 80% from the unique CDR3 sequences shared by the 3 rd and 3.1 rd generation test end products.

Percentage of unique CDR3 sequences shared between L4064 control and TIL cell products collected from 3.1 generation test: 2163 sequences were shared between the 3 rd and 3.1 rd generation test end products, corresponding to 87% of the first 80% from the unique CDR3 sequences shared by the 3 rd and 3.1 rd generation test end products.

Collect 1×10 collected from day 16 of different processes ⁶ Number of unique CD3 sequences identified by individual cells. Based on the number of unique peptide CDRs in the sample, the 3.1 generation test conditions showed slightly higher clonal diversity compared to 3.0 generation.

Shannon entropy diversity index is a more reliable and common comparative measure, since both tumors show slightly higher diversity in the 3.1 rd generation than in the 3 rd generation, suggesting that the 3.1 rd generation test conditions TCR vβ reservoirs are more polyclonal than in the 3.0 rd generation.

In addition, the TCR vβ reservoirs of the 3.1 generation test conditions for both tumors L4063 and L4064 showed more than 87% overlap with the corresponding reservoir of the 3.0 generation procedure.

The value of IL-2 concentration of the used medium for test L4064 at 3.1 generation on reactivation day was lower than expected (similar to the 3.1 generation control and 3.0 generation conditions).

The low value may be due to pipetting errors, but because the least sample is collected, it is not possible to repeat the assay.

Spent medium from sample L4064 on day 10/11 of the longitudinal scale-up was not collected and was not included in the supernatant IL-2 concentration analysis and metabolite analysis.

Conclusion(s). The 3.1 generation test conditions including feeder cells and OKT-3 at day 0 showed higher TVC cell doses at day 16 of collection compared to the 3.0 generation and 3.1 generation controls. The final product of the 3.1 rd generation test conditions has a TVC that is about 2.5 times higher than the 3.0 rd generation.

The 3.1 generation test conditions with OKT-3 and feeder cells added on day 0 reached the maximum capacity of the flask when both tumors L4063 and L4064 were collected. Under these conditions, if a maximum of 4 flasks were started on day 0, the final cell dose might be between 80 and 100X 10 ⁹ Between TILs.

All quality attributes of the final TIL product, such as phenotyping including purity, depletion, activation and memory signature, were maintained and comparable between the 3.1 generation test and 3.0 generation process. Telomere length of the TIL end product and IL-2 consumption of used medium are comparable between the 3.0 rd and 3.1 rd generation processes.

In both tumors analyzed, IFN-. Gamma.production of the final TIL product of passage 3.1 with feeder cells and OKT-3 added on day 0 was 3-fold higher than passage 3.0, suggesting that passage 3.1 process produced an effective TIL product.

No difference in the amount of glucose or lactate between the test conditions was observed. No difference in glutamine and ammonia was observed between the 3.0 th and 3.1 th passages between the medium conditions. The low amount of glutamine in the medium does not limit cell growth and implies that the addition of only GlutaMax to the medium is sufficient to give the nutrients needed to proliferate the cells.

The scale up day of L4063 and L4064 did not show significant differences in cell numbers reached on day 11 and day 10, respectively, and with respect to the day of process collection, the metabolite consumption of both was comparable during the whole process. This observation suggests that the 3.0 generation optimization process may be flexible over the treatment day, thereby facilitating flexibility in the manufacturing schedule.

The 3.1 generation procedure with feeder cells and OKT-3 added on day 0 showed higher clonal diversity as measured by CDR3TCRab sequence analysis compared to 3.0 generation.

Fig. 32 depicts an embodiment of the 3 rd generation process (3 rd generation optimization process). The 3 rd generation optimization procedure TIL amplification can use standard medium and CTS Optimizer serum-free medium. Taking CTS Optimizer serum-free medium as an example, it is recommended to increase the GlutaMax of the medium to a final concentration of 4mM.

The feasibility of all study conditions has been established in all experiments. All experiments were performed with the same batches of key raw materials such as IL-2, human serum-AB, allogeneic feeder cells, OKT-3 in all experiments and conditions and between donor tumor tissues.

Comparability was judged by the ability of any of the groups studied to meet clinical product release criteria according to previous specifications for TIL products cryopreserved on day 22.

Example 12: tumor amplification procedure using defined media

The above disclosed processes may be performed, including the 2 nd and 3 rd generation processes, but with the CM1 and CM2 medium replaced with defined medium (e.g., CTS ^TM OpTmizer ^TM T cell expansion SFM, thermoFisher, including, for example, DM1 and DM 2).

Example 13: exemplary production of cryopreserved TIL cell therapies

This example describes an exemplary cGMP manufacture of TIL cell therapy in G-Rex flasks (or alternatively, gas permeable bags) according to current pharmaceutical good manufacturing practices.

Table 80: procedure augmentation exemplary plan

Table 81: culture flask volume

Type of flask	Working volume/bottle (mL)
		G-Rex 100MCS	1000
G-Rex 500MCS	5000

Preparation of CM1 Medium on day 0. In BSC, reagents were added to RPMI 1640 medium flasks. The following reagents were added: thermally deactivating human AB serum (100.0 mL); glutaMax (10.0 mL); gentamicin sulfate 50mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL).

The optional material is removed from the BSC. The media reagent in the BSC was transferred out leaving gentamicin sulfate and HBSS in the BSC for formulation of the wash media preparation.

An aliquot of IL-2 was thawed. A1.1 mL aliquot of IL-2 (6X 10) ⁶ IU/mL) (BR 71424) until all ice is melted. IL-2 lot number and expiration date were recorded.

IL-2 stock was transferred to medium. In BSC, 1.0mL of IL-2 stock was transferred to prepared CM1 day 0 medium flasks. 1 bottle of CM1 day 0 Medium and IL-2 (6X 10) ⁶ IU/mL)1.0mL。

The G-Rex100MCS is delivered into the BSC. The G-Rex100MCS (W3013130) is delivered aseptically into the BSC.

All complete CM1 day 0 media was pumped into G-Rex100MCS flasks (tissue fragment conical tubes or GRex100 MCS).

Preparation of tumor washing Medium on day 0. In BSC, 5.0mL gentamicin (W3009832 or W3012735) was added to a 1X 500mL HBSS culture medium (W3013128) bottle. Each bottle is added with: HBSS (500.0 mL); gentamicin sulfate 50mg/mL (5.0 mL). The prepared gentamicin-containing HBSS was filtered through 1l of 0.22 micron filter units (W1218810).

Tumor treatment on day 0. Tumor samples were obtained and immediately transferred to a kit for treatment at 2 to 8 ℃.

Tumor wash medium was aliquoted. Tumor wash 1 was performed using 8 "forceps (W3009771). Tumors were removed from the sample vials and transferred to prepared "wash 1" dishes. Tumor wash 2 and tumor wash 3 were then performed.

Tumors were measured and evaluated. It was evaluated whether >30% of the total tumor area was observed as necrotic and/or adipose tissue. The segmentation (if applicable) is cleared. If the tumor is large and >30% of the tissue exterior is necrotic/adipose, a "clean-up split" is performed by removing the necrotic/adipose tissue while preserving the tumor internal structure using a combination of scalpels and/or forceps.

The tumor is segmented. The tumor sample is cut into uniform, appropriately sized pieces (up to 6 intermediate pieces) using a combination of scalpels and/or forceps. Intermediate tumor fragments were metastasized. Dividing tumor fragments into large fragments a sheet of about 3 x 3mm in size. The intermediate fragments were stored to prevent drying.

The middle segment segmentation is repeated. The number of pieces collected was measured. If the desired tissue is still present, additional preferred tumor pieces are selected from the "preferred intermediate segment" 6-well plate to fill the droplets with up to 50 pieces.

Preparing a conical tube. The tumor pieces were transferred to 50mL conical tubes. BSC was prepared 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. Tumor pieces are uniformly distributed.

The G-Rex100MCS was incubated with the following parameters: incubation methodG-Rex culture flask: temperature LED display: 37.0+/-2.0 ℃; CO ₂ The percentages are as follows: 5.0.+ -. 1.5% CO ₂ . And (3) calculating: incubation time; lower limit = incubation time +252 hours; upper limit = incubation time +276 hours.

After the process has been completed, any remaining warm medium and thawed IL-2 aliquots are lost.

Day 11-Medium preparation. The incubator was monitored. Incubator parameters: temperature LED display: 37.0+/-2.0 ℃; CO ₂ The percentages are as follows: 5.0.+ -. 1.5% CO ₂ 。

3 1000mL RPMI 1640 medium (W3013112) flasks and 3 1000mL AIM-V (W3009501) flasks were warmed in the incubator for 30 min or more. RPMI 1640 medium was removed from the incubator. RPMI 1640 medium was prepared. The medium was filtered. Thawing 3 1.1mL aliquots of IL-2 (6X 10) ⁶ IU/mL) (BR 71424). AIM-V medium was removed from the incubator. IL-2 was added to AIM-V. The 10L Labtainer bag and the relay pump transfer set were aseptically transferred to the BSC.

10L Labtainer media bags were prepared. Baxa pumps were prepared. 10L Labtainer media bags were prepared. The medium was pumped into a 10L Labtainer. The pump (pumpmatic) was removed from the Labtainer bag.

The medium was mixed. The bag was gently massaged for mixing. The medium was sampled according to the sample plan. 20.0mL of medium was removed and placed in a 50mL conical tube. A cell count dilution tube was prepared. In BSC, 4.5mL of AIM-V medium labeled "for cell count dilution" and lot number was added to four 15mL conical tubes. The reagent was transferred from BSC to 2 to 8 ℃. 1L transfer packs were prepared. Outside the BSC, 1L transfer packets were joined (per process notice 5.11) to a transfer set attached to a prepared "complete CM2 day 11 media" bag. Feeder cell transfer bags were prepared. Complete CM2 day 11 medium was incubated.

Day 11-TIL Collection. Pretreatment table. Incubator parameters: temperature LED display: 37.0+/-2.0 ℃; CO ₂ The percentages are as follows: 5.0.+ -. 1.5% CO ₂ . The G-Rex100MCS was removed from the incubator. Prepare 300mL transfer bags. The transfer packet is spliced to the G-Rex100MCS.

The flask for TIL collection was prepared and TIL collection was initiated. TIL collection. Using GatheRex, the cell suspension was transferred through a hemofilter into a 300mL transfer bag. The membrane was inspected for presence of adherent cells.

Rinsing the culture bottle membrane. The tube clamp on G-Rex100MCS was closed. Ensure that all clamps are closed. Heat seal TIL and "supernatant" transfer packets. The TIL suspension volume was calculated. Supernatant transfer packs were prepared for sampling.

Bac-T samples were withdrawn. In the BSC, about 20.0mL of supernatant was withdrawn from the 1L "supernatant" transfer packet and dispensed into a sterile 50mL conical tube.

BacT was inoculated according to the sample schedule. 1.0mL of sample was removed from the prepared 50mL conical tube labeled BacT using an appropriately sized syringe and inoculated into an anaerobic jar.

Incubating the TIL. The TIL transfer packs were placed in an incubator until needed. Cell counting and calculation were performed. The average viable cell concentration and viability of the cell counts performed were determined. Viability ≡2. Viable cell concentration ≡2. The upper and lower counts were determined. Lower limit: average viable cell concentration x 0.9. Upper limit: average viable cell concentration x 1.1. Confirm that both counts are within acceptable limits. The average viable cell concentration of all four counts performed was determined.

Adjusting the volume of the TIL suspension: after removal of the cell count sample, the adjusted volume of the TIL suspension was calculated. Total TIL cell volume (a). The sample volume (4.0 mL) was counted for the cells removed (B). Adjusted total TIL cell volume c=a-B.

Total surviving TIL cells were calculated. Average viable cell concentration: total volume; total surviving cells: c=a×b.

Flow cytometry calculation: if the total surviving TIL cell count is not less than 4.0x10 ⁷ The volume is calculated to obtain 1.0X10 of flow cytometry samples ⁷ Individual cells.

Total surviving cells required for flow cytometry: 1.0X10 ⁷ Individual cells. Cell volume required for flow cytometry: viable cell concentration divided by 1.0X10 ⁷ And (3) cells A.

Calculation is equal to 2.0X10 ⁸ TIL suspension volumes of individual surviving cells. If necessary, the excess volume of TIL cells to be removed is calculated and the excess TIL is removed and optionally placed in an incubator. The total excess TIL removed is calculated as needed.

The amount of CS-10 medium to be added to the excess TIL cells was calculated, wherein the target cell concentration for freezing was 1.0X10 ⁸ Individual 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 residual 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.

Calculation to obtain 1X 10 for cryopreservation ⁷ Cell volume required for individual cells. Samples for cryopreservation were removed. The TIL was placed in an incubator.

Cryopreserved samples. The conical tube was observed and CS-10 was slowly added and the 0.5mL CS10 volume added was recorded.

Day 11 feeder cells. Feeder cells were obtained. 3 bags of at least two different lot number feeder cells were obtained from LN2 freezer. Cells were kept on dry ice until ready for thawing. A water bath or Cryotherm was prepared. The feeder cells were thawed in a water bath or Cytotherm at 37.0.+ -. 2.0 ℃ for about 3 to 5 minutes 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 pack. Feeder cells were dispensed from the syringe into transfer packs. Pooled feeder cells were mixed and labeled for transfer packs.

The total volume of feeder cell suspension in the transfer bag was calculated. The cell count samples were removed. A separate 3mL syringe was used for each sample, and a 4X 1.0mL cell count sample was withdrawn from the feeder cell suspension transfer bag using a needleless injection port. Each sample was aliquoted into labeled frozen vials. Cell counts were performed and multiplication factors were determined, selected protocols and input. The average viable cell concentration and viability of the cell counts performed were determined. 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. After removal of the cell count sample, the adjusted volume of feeder cell suspension was calculated. Total surviving feeder cells were 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 clamp chain bag and thawed in a 37.0.+ -. 2.0 ℃ water bath or Cytotherm for about 3 to 5 minutes, 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 pack.

Dilutions were made as needed, adding 4.5mL AIM-V medium to four 15mL conical tubes. Cell counts were prepared. A separate 3mL syringe was used for each sample and a 4 x 1.0mL cell count sample was removed from the feeder cell suspension transfer bag using a needleless injection port. Cell counting and calculation were performed. The average viable cell concentration of all four counts performed was determined. The volume of the feeder cell suspension was adjusted and after removal of the cell count sample, the adjusted volume of the feeder cell suspension was calculated. Total feeder cell volume minus 4.0mL removed. Calculation to obtain 5×10 ⁹ Volume of feeder cell suspension required for individual surviving feeder cells. 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 of OKT3 was withdrawn and OKT3 was added. The feeder cell suspension transfer bag was heat sealed.

On day 11, G-Rex fill and inoculate, set G-Rex500MCS. The "complete CM2 day 11 medium" was removed from the incubator and the medium was pumped into G-Rex500MCS. 4.5L of medium was pumped into the G-Rex500MCS, filled to the line indicated by the flask. The flasks were heat sealed and incubated as necessary. Feeder cell suspension transfer packs were conjugated to G-Rex500MCS. Feeder cells were added to G-Rex500MCS. And (5) heat sealing. The TIL suspension transfer bag was attached to a flask. TIL is added to G-Rex500MCS. And (5) heat sealing. At 37.0+ -2.0deg.C, CO ₂ The percentages are as follows: 5.0.+ -. 1.5% CO ₂ The G-Rex500MCS was incubated.

And calculating the incubation window. Calculations were made to determine the appropriate time to move the G-Rex500MCS out of the incubator on day 16. Lower limit: incubation time +108 hours. Upper limit: incubation time +132 hours.

On day 11, excess TIL was cryopreserved. The method is feasible: excess TIL vials were frozen. Verify that CRF has been set prior to freezing. Freezing and preserving. Vials were transferred from the controlled speed freezer to 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, culture medium preparation. AIM-V medium was pre-warmed. The time was calculated to warm the media of media bags 1, 2 and 3. Ensure that all bags have been warmed for a period of between 12 and 24 hours. 10L Labtainer was set for the supernatant. The larger diameter end of the fluid pump transfer set was attached to one female port of a 10L Labtainer bag using a luer fitting. 10L Labtainer was set for the supernatant and labeled. 10L Labtainer was set for the supernatant. Before removal from the BSC, all clamps are ensured to be closed. Note that: the supernatant bag was used during TIL collection, which can be performed simultaneously with the media preparation.

Thawing IL-2. 5 1.1mL aliquots of IL-2 (6X10) were thawed per bag of CTS AIM V medium ⁶ IU/mL) (BR 71424) until all ice melts. 100.0mL of GlutaMax was aliquoted. IL-2 was added to GlutaMax. CTS AIM V media bags were prepared for formulation. CTS AIM V media bags were prepared for formulation. And (5) erecting a Baxa pump. The preparation medium was prepared. GlutaMax+IL-2 was pumped to the bag. Monitoring parameters: temperature LED display: 37.0+ -2.0 ℃, CO ₂ The percentages are as follows: 5.0.+ -. 1.5% CO ₂ . The complete CM4 day 16 medium was warmed. Preparing dilution.

Day 16 REP split bottle. Monitoring incubator parameters: temperature LED display: 37.0+ -2.0 ℃, CO ₂ The percentages are as follows: 5.0.+ -. 1.5% CO ₂ . The G-Rex500MCS was removed from the incubator. Prepare and mark 1L transfer ladle as TIL suspension and weigh 1L.

The volume of the G-Rex500MCS decreases. About 4.5L of the culture supernatant was transferred from G-Rex500MCS to 10L Labtainer.

Flasks were prepared for TIL collection. After removal of the supernatant, all clamps to the red line were closed.

Initial TIL Collection. The flask was vigorously tapped and the medium vortexed to release the cells, ensuring that all cells had been detached.

TIL collection. All clamps leading to the TIL suspension transfer bag were released. Cell suspensions were transferred to TIL suspension transfer bags using GatheRex. Note that: the maintenance of the beveled edge was determined until all cells and medium had been collected. The membrane was inspected for presence of adherent cells. Rinsing the culture bottle membrane. The tube clamp on G-Rex500MCS was closed. The transfer bag containing the TIL was heat sealed. 10L Labtainer containing the supernatant was heat sealed. The weight of the transfer bag containing the cell suspension was recorded and the volume suspension calculated. Transfer packs were prepared for sample removal. The test sample of cell supernatant was removed.

Sterility of the product&BacT test sample. 1.0mL of sample was removed from the prepared 15mL conical tube labeled BacT. The cell count samples were removed. In the BSC, 4X 1.0mL of cell count samples were removed from the TIL suspension transfer pack using a separate 3mL syringe for each sample.

Remove mycoplasma samples. Using a 3mL syringe, 1.0mL was removed from the TIL suspension transfer bag and placed into a 15mL conical tube labeled "mycoplasma diluent".

Transfer packs were prepared for inoculation. The TIL was placed in an incubator. The cell suspension was removed from the BSC and placed in an incubator until needed. Cell counting and calculation were performed. The cell count samples were initially diluted by adding 0.5mL of cell suspension to 4.5mL of AIM-V medium prepared, which gave 1: and (5) diluting by 10. The average viable cell concentration and viability of the cell counts performed were determined. The upper and lower counts were determined. Note that: dilution can be adjusted according to the desired cell concentration. The average viable cell concentration of all four counts performed was determined. The volume of the TIL suspension was adjusted. After removal of the cell count sample, the adjusted volume of the TIL suspension was calculated. Total TIL cell volume minus 5.0mL removed for testing.

Total surviving TIL cells were calculated. The total number of flasks to be inoculated was calculated. Note that: the maximum number of G-Rex500MCS flasks to be inoculated was 5. If the calculated number of flasks to be inoculated exceeds five, only five flasks are inoculated using the entire volume of available cell suspension.

The number of flasks for the subculture was counted. The number of bags of medium required in addition to the bags prepared was calculated. One "CM4 day 16 medium" 10L bag was prepared for every two G-Rex-500M flasks calculated as required. Proceed to inoculate the first GREX-500M flask while additional medium is prepared and warmed. The calculated number of additional medium bags determined were prepared and warmed. G-Rex500MCS is filled. Preparation of pumped Medium and pumping of 4.5L of Medium into G-Rex500MCS. And (5) heat sealing. And (5) repeating filling. Incubate the flask. The target volume of TIL suspension to be added to the new G-Rex500MCS flask was calculated. If the calculated number of flasks exceeds five, only five flasks are inoculated with the entire volume of the cell suspension. Flasks were prepared for inoculation. The G-Rex500MCS was removed from the incubator. The G-Rex500MCS was prepared for pumping. All clamps were closed except for the large filter line. The TIL was removed from the incubator. Cell suspensions were prepared for inoculation. The "TIL suspension" transfer bag was aseptically joined (according to process notice 5.11) to the pump inlet line. The TIL suspension bag was placed on a scale.

The flasks were inoculated with the TIL suspension. The calculated volume of TIL suspension was pumped into the flask. And (5) heat sealing. Fill the remaining flask.

The incubator was monitored. Incubator parameters: temperature LED display: 37.0+ -2.0 ℃, CO ₂ The percentages are as follows: 5.0.+ -. 1.5% CO ₂ . Incubate the flask.

The time frame for moving the G-Rex500MCS out of the incubator on day 22 was measured.

Day 22 wash buffer preparation. A10L Labtainer bag was prepared. In the BSC, a 4 "plasma transfer set was attached to a 10L Labtainer bag via a luer connection. A10L Labtainer bag was prepared. All clamps are closed before switching out of the BSC. Note that: A10L Labtainer bag was prepared for each two G-Rex500MCS flasks that were collected. Pump Plasmalyte to 3000mL bag, let throughAir was removed from the 3000mL origin bag by reversing the pump and operating the bag position. A bag of 25% to 3000mL of human albumin was added. A final volume of 120.0mL of human albumin of 25% was obtained.

IL-2 diluent was 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 withdrawn from the needleless injection port.

Thawing a 1.1mL of IL-2 (6X 10) ⁶ IU/mL) until all ice melts. 50. Mu.L of IL-2 stock solution (6X 10) ⁶ IU/mL) to a 50mL conical tube labeled "IL-2 diluent".

Cryopreservation formulations. The 5 frozen cassettes were pre-treated at 2 to 8 ℃ for final product cryopreservation.

Cell count dilutions were prepared. In BSC, 4.5mL of the indicated lot number and AIM-V medium for "cell count dilution" were added to 4 separate 15mL conical tubes. Cell counts were prepared. The 4 frozen vials were labeled vial numbers (1 to 4). The vial is kept in the BSC for use.

Day 22 TIL Collection. 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. TIL collection bags were prepared and labeled. And sealing the redundant connection. Volume reduction: about 4.5L of supernatant was transferred from G-Rex500MCS to the supernatant bag.

Flasks were prepared for TIL collection. Collection of TIL was initiated. The flask was vigorously patted and the medium swirled to release the cells. Ensure that all cells have been exfoliated. Initial TIL collection. All clamps leading to the TIL suspension collection bag were released. TIL collection was performed. The TIL suspension was transferred to a 3000mL collection bag using GatheRex. The membrane was inspected for presence of adherent cells. Rinsing the culture bottle membrane. The jaws on the G-Rex500MCS are closed and all jaws are ensured to be closed. The cell suspension was transferred to a LOVO source bag. Closing all clamps. And (5) heat sealing. Remove 4X 1.0mL of cell count sample.

Cell counting was performed. Cell counting and calculation was performed using NC-200 and procedure notes 5.14. The cell count samples were initially diluted by adding 0.5mL of cell suspension to 4.5mL of AIM-V medium prepared. This gives 1: and (5) diluting by 10. The average viability, viable cell concentration and total nucleated cell concentration of the cell counts performed were determined. The upper and lower counts were determined. The average viability, viable cell concentration and total nucleated cell concentration of the cell counts performed were determined. Weigh the LOVO source bag. Total surviving TIL cells were calculated. The total nuclear cells were counted.

Preparing mycoplasma diluent. Remove 10.0mL from one supernatant bag through the luer sample port and place into a 15mL conical tube.

The "TIL G-Rex collection" protocol was performed to determine the final product target volume. And (5) loading a disposable kit. The filtrate bag was removed. The filtrate volume was input. The filtrate container is placed on a table. Plasmalyte was attached. Plasmalyte was verified as attached and was observed to be moving. The source container is attached to the tubing and the source container is verified as attached. Confirm that PlasmaLyte is moving.

Final formulation and filling. Target volume/bag calculation. The volumes of CS-10 and LOVO wash buffer were calculated to formulate blank bags. CRF blanks were prepared.

The volume of IL-2 to be added to the final product was calculated. The final IL-2 concentration (IU/mL) required is 300IU/mL. IL-2 working stock: 6X 10 ⁴ IU/mL. And assembling the connecting device. The 4S-4M60 was aseptically conjugated to CC2 cell ligation. The CS750 freezer bag is aseptically joined to the prepared tooling. The CS-10 bag was joined to the 4S-4M60 puncture needle. Preparing the IL-2-containing TIL. From "IL-2 6X 10" using a syringe of appropriate size ⁴ "aliquots removed the measured amount of IL-2. The formulated TIL bag is labeled. The formulated TIL bag was added to the apparatus. CS10 was added. The syringe is replaced. About 10mL of air was drawn into the 100mL syringe and the 60mL syringe on the device was replaced. CS10 was added. CS-750 bags were prepared. Cells were dispensed.

The air in the final product bag was removed and the retentate was removed. Once the final end product bag has been filled, all clamps are closed. About 10mL of air was drawn into a new 100mL syringe and the syringe on the device was replaced. The reservations were dispensed into 50mL conical tubes and the tubes were labeled "reserved" and lot number. Each bag repeats the air removal step.

The final product was prepared for cryopreservation, including visual inspection. The freezer bag is kept on ice bags or at 2 to 8 ℃ until cryopreservation.

The cell count samples were removed. Using a pipette of appropriate size, 2.0mL of the retentate was removed and placed into a 15mL conical tube for cell counting. Cell counting and calculation were performed. Note that: only one sample was diluted to the proper dilution to verify that the dilution was sufficient. Additional samples were diluted to the appropriate dilution factor and counted. The average viable cell concentration and viability of the cell counts performed were determined. The upper and lower counts were determined. Note that: dilution can be adjusted according to the desired cell concentration. A calculated IFN- γ sample. Heat sealing the final product bag.

Samples were identified and collected according to the exemplary sample plan in table 82.

Table 82: sample planning

Sample of	Number of containers	Sample volumes added to each	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 reservation	4	1.0mL	2mL frozen vials
Satellite vial	10	0.5mL	2mL frozen vials

Sterility and BacT test. And (5) testing and sampling. In the BSC, 1.0mL of sample was removed from the collected retained cell suspension using a syringe of appropriate size and inoculated into an anaerobic jar. The above was repeated for the aerobic flask.

Freezing and preserving the final product. A Controlled Rate Freezer (CRF) was prepared. Verify that CRF has been set and set CRF probe. The final product and sample were placed in CRF. The time required to reach 4 ℃ + -1.5 ℃ was determined and CRF run was performed. CRF is completed and stored. After the run is completed, the CRF is stopped. The cassette and vial are removed from the CRF. Transfer of cassettes and vials to gas phase LN ₂ And (5) storing. The storage location is recorded.

Post-processing and analysis of the final drug product included the following tests: (day 22) cd3+ cells of day 22 REP as determined by flow cytometry; (day 22) gram stain method (GMP); (day 22) bacterial endotoxin test (GMP) by gel LAL assay; (day 16) BacT sterility assay (GMP); (day 16) detection of mycoplasma DNA (GMP) by TD-PCR; acceptable appearance attributes; (day 22) BacT sterility assay (GMP) (day 22); (day 22) IFN-gamma assay. Other potency assays described herein are also employed to analyze TIL products.

Example 14: multicenter study of autologous tumor infiltrating lymphocytes in stage 2 of solid tumor patients

Overview of study design. This example describes one prospective, open label, multiple study cohorts, non-randomized group, multiple center phase 2 study that used TIL in combination with pembrolizumab or TIL as monotherapy and TIL prepared as described herein and in this example.

Purpose(s). The main objective was to evaluate the efficacy of a combination of autologous TIL with pembrolizumab in MM, HNSCC or NSCLC patients or TIL as monotherapy in recurrent or refractory (r/r) NSCLC patients who had previously progressed on or after CPI treatment as judged by researchers evaluating Objective Response Rate (ORR) using the solid tumor response evaluation criteria (RECIST 1.1).

Identification of the safety profile of combination of TIL with pembrolizumab in MM, HNSCC and NSCLC patients or TIL as monotherapy in r/r NSCLC patients, as measured by the incidence of ≡3 treatment-induced adverse events (TEAEs).

The secondary objective was to further evaluate the efficacy of autologous TIL in combination with pembrolizumab in MM, HNSCC and NSCLC patients or TIL as monotherapy in r/rnnsclc patients using Complete Response (CR) rate, duration of response (DOR), disease Control Rate (DCR), progression Free Survival (PFS) (assessed by the investigator using RECIST 1.1) and Overall Survival (OS).

The study included the following study cohorts:

study cohort 1A: the combination of TIL therapy with pembrolizumab was used in patients who were unresectable or MM at stage IIIC or IV and received +.3 previous on-line systemic therapies excluding immunotherapy. If previously treated, the patient must have a radiological documented progression at or after the last treatment.

Study cohort 2A: the combination of TIL therapy with pembrolizumab is used in patients with advanced, recurrent or metastatic HNSCC (e.g., stage T1N1-N2B, stage T2-4N 0-N2B) and who receive ∈3 previous online systemic therapies excluding immunotherapy. If previously treated, the patient must have a radiological documented progression at or after the last treatment.

Study cohort 3A: the combination of TIL therapy with pembrolizumab was used for patients with locally advanced or metastatic (stage III to IV) NSCLC and received ∈3 previous online systemic therapies excluding immunotherapy. If previously treated, the patient must have a radiological documented progression at or after the last treatment.

Study cohort 3B: TIL therapy is used as a single dose for stage III or IV NSCLC and previously received CPI systemic therapy (e.g., anti-PD-1/anti-PD-L1) as part of ∈3 of the previous online systemic therapies. If previously treated, the patient must have a radiological documented progression at or after the last treatment.

Patients on study cohorts 3A and 3B (NSCLC) with available oncogene-driven tumors for effective target therapies must have received at least one on-line target therapy.

All patients received autologous cryopreserved TIL therapy (whether pembrolizumab or not, depending on study cohort allocation) and previously received a non-myeloablative lymphocyte depletion (NMA-LD) pretreatment regimen consisting of cyclophosphamide and fludarabine. Following TIL infusion, up to 6 doses of IV interleukin-2 (IL-2) are administered.

The following routine study period occurred in all 4 study cohorts unless otherwise indicated.

Screening and tumor excision: up to 4 weeks (28 days) after study entry; production of TIL product: about less than or equal to 22 days after tumor resection; and the treatment period as discussed below.

Treatment period (study queues 1A, 2A and 3A): up to 2 years, including NMA-LD (7 days), TIL infusion (1 day) and subsequent IL-2 administration (1 to 4 days). Patients received a single infusion of pembrolizumab after completing tumor resection for TIL production and baseline scan but before initiating an NMA-LD regimen. The next dose of pembrolizumab will be no earlier than after completion of IL-2, after which Q3w±3 days are continued for no more than 2 years (24 months) or until disease progression or unacceptable toxicity (based on the first occurrence). End of treatment (EOT) palpation occurs within 30 days after the last dose of pembrolizumab. This return diagnosis may be combined with an end of assessment (EOA) return diagnosis, if applicable (e.g., pembrolizumab discontinuation occurs at the onset of disease progression or new anti-cancer therapy).

Treatment period (study cohort 3B): up to 12 days, including NMA-LD (7 days), TIL infusion (1 day) and subsequent IL-2 administration (1 to 4 days). EOT back diagnosis occurs after the patient receives the last dose of IL-2. EOT back diagnosis is performed within 30 days after treatment discontinuation and may be combined with any scheduled back diagnosis occurring during this interval during the evaluation period.

Evaluation period: beginning and ending after day 0 TIL infusion at disease progression, new anti-cancer therapy onset, partial withdrawal study evaluation consent, or at 5 years (month 60) based on the pre-emergence. End of assessment (EOA) back diagnosis occurs after the patient reaches disease progression or begins a new anti-cancer therapy.

TIL autotherapy using TIL prepared as described herein comprises the steps of:

1) Tumor excision to provide autologous tissue that serves as a source of TIL cellular products;

2) Producing TIL products at a central Good Manufacturing Practice (GMP) agency;

3) 7 days NMA-LD pretreatment protocol;

4) Study queues 1A, 2A and 3A: patients received a single infusion of pembrolizumab after completion of tumor resection and baseline scan for TIL production but before initiating an NMA-LD regimen. The next dose of pembrolizumab will be no earlier than after IL-2 is completed, after which it lasts Q3w±3 days.

5) Infusion of autologous TIL product (day 0); and

6) IV IL-2 is administered up to 6 doses.

In study cohorts 1A, 2A and 3A, the next dose of pembrolizumab was not earlier than after IL-2 was completed, after which Q3 W.+ -. 3 days for +.2 years (24 months) or until disease progression or unacceptable toxicity (based on the preexisting).

The flow chart of study queues 1A, 2A and 3A can be seen in FIG. 7. The flow chart of study queue 3B can be seen in fig. 8. Patients were assigned to appropriate study cohorts for tumor indications.

TIL therapy+pembrolizumab (study queues 1A, 2A and 3A). Patients were screened and scheduled for tumor resection. The patient is then resected from more than one tumor lesion and sent to a central manufacturing facility for TIL production.

Next, NMA-LD protocol was initiated and consisted of 2 days IV cyclophosphamide (60 mg/kg) plus mesna (mesna) (care per center standard or USPI/SmPC) on day-7 and day-6 followed by 5 days IV fludarabine (25 mg/m) ² : day-5 to day-1).

Patients in study cohorts 1A, 2A and 3A received a single infusion of pembrolizumab after completion of tumor resection and baseline scan for TIL production and prior to initiation of the NMA-LD regimen. IL-2 was administered at a dose of 600,000IU/kg IV, beginning as soon as 3 hours but no later than 24 hours after completion of the TIL infusion on day 0. Additional IL-2 administration will be administered about once every 8 to 12 hours, up to 6 doses. The second dose of pembrolizumab is not earlier than after completion of IL-2. Prior to the second pembrolizumab administration, the patient should recover from all IL-2-associated toxicities (grade ∈2). Pembrolizumab will thereafter last for Q3 W+ -3 days for < 2 years (24 months) or until disease progression or unacceptable toxicity (based on the first occurrence).

TIL therapy as a single agent (study cohort 3B). Patients were screened and scheduled for tumor resection. The patient is then resected from more than one tumor lesion and sent to a central manufacturing facility for TIL production.

Next, NMA-LD regimen consisted of 2 days IV cyclophosphamide (60 mg/kg) plus mesna (mesna) (care per center standard or USPI/SmPC) on day-7 and day-6 followed by 5 days IV fludarabine (25 mg/m) ² : day-5 to day-1).

Infusion of tumor-derived autologous TIL product occurs no earlier than 24 hours after the last dose of fludarabine. IL-2 was administered at a dose of 600,000IU/kg IV, beginning as soon as 3 hours after completion of TIL infusion but not later than 24 hours.

Additional IL-2 administration is about once every 8 to 12 hours, up to 6 doses.

Production and expansion of tumor infiltrating lymphocytes. The TIL autologous cellular products consist of viable cytotoxic T lymphocytes derived from the patient's tumor/lesion, which are manufactured ex vivo at a central GMP facility. For example, an exemplary flowchart depicting a TIL generation process is provided in fig. 9.

The TIL manufacturing process starts after surgical excision of each primary or secondary metastatic tumor lesion with a diameter of 1.5cm or more in a patient at the clinical center. Multiple tumor lesions from various anatomical locations can be resected as total tumor tissue total aggregates; however, the aggregate should not exceed a diameter of 4.0cm or weigh 10g because of the limited amount of biological preservation medium present in the transport bottle.

Once the tumor lesions are placed in the biological preservation transport bottle, they are transported to a central GMP manufacturing facility at 2 ℃ to 8 ℃ using express delivery. Upon arrival, tumor samples were fragmented and then incubated with human recombinant IL-2 for about 11 days prior to the rapid amplification protocol (pre-REP).

These REP pre-cells were then further expanded using the Rapid Expansion Protocol (REP) for 11 days in the presence of IL-2, OKT3 (murine anti-human CD3 monoclonal antibody, also known as [ moruzumab-CD 3 ]), and irradiated allogeneic Peripheral Blood Mononuclear Cells (PBMCs) as feeder cells.

The expanded cells are then collected, washed, formulated, cryopreserved and shipped to a clinical center by express delivery. The dosage form of the TIL cellular product is a cryopreserved autologous "live cell suspension" that is ready for infusion into patients from which the TIL is derived. The patient expects to receive a complete dose of the product, which, according to the product specifications, contains between 1 x 10 ⁹ And 150X 10 ⁹ Surviving cells between individuals. Clinical experience has shown that objective tumor responses can be achieved within this dose range, and also have been shown to be safe (Radvanyi et al, clin Cancer res.2012,18,6758-70). The complete dose of product is provided in up to four infusion bags.

Preparation of patients to receive TIL cellular products. The NMA-LD pretreatment regimen used in this study (i.e., 2 days cyclophosphamide plus mesna followed by 5 days fludarabine) was a method based on the U.S. national cancer institute (National Cancer Institute) development and testing. Rosenberg et al Clin.cancer Res.2011,17 (13), 4550-7; radvanyi et al, clin.cancer res.2012,18 (24), 6758-70; dudley et al, J.Clin.Oncol.2008,26 (32), 5233-9; pilon-Thomas et al, J.Immunother.2012,35 (8), 615-20; dudley et al, J.Clin.Oncol.2005,23 (10), 2346-57; and Dudley et al, science 2002,298 (5594), 850-4. After a 7 day pretreatment regimen, the patient infuses TIL cell-based product.

IV IL-2 (600,000IU/kg) was administered once every 8 to 12 hours after TIL infusion, with the first dose administered between 3 to 24 hours after TIL infusion was completed and lasting up to 6 doses at maximum. The dose of IL-2 can be calculated according to the actual body weight, according to institutional standards.

The patient population for each study cohort was selected as follows:

study cohort 1A: patients had a definite unresectable MM (stage IIIC or IV, histologically confirmed by the united states joint committee for cancer [ AJCC ] staging system). Patients with ocular melanoma are excluded. Patients previously had not received an immune tumor targeting agent. If the BRAF mutation is positive, the patient may previously receive BRAF/MEK target therapy.

Study cohort 2A: patients have advanced, recurrent and/or metastatic HNSCC and may be untreated; pathological reporting of histological diagnosis of the primary tumor is necessary. Patients previously had not received an immunotherapy regimen.

Study cohort 3A: patients have established stage III or IV NSCLC (squamous, adenocarcinoma, large cell carcinoma). Patients with oncogene driven tumors for which effective target therapies are available have received at least one on-line target therapy.

Study cohort 3B: patients have diagnosed stage III or IV NSCLC (squamous, adenocarcinoma, large cell carcinoma) and have previously received CPI systemic therapy (e.g., anti-PD-1/anti-PD-L1). Patients with oncogene driven tumors for which effective target therapies are available have received at least one on-line target therapy.

All patients had received up to 3 previous systemic anti-cancer therapies (see inclusion criteria below), but the immunotherapy for study cohorts 1A, 2A and 3A was excluded. If previously treated, the patient has radiation-confirmed progress at or after the last treatment.

Inclusion criteria. Patients must meet all of the following inclusion criteria to participate in the study:

1) All patients had their respective histological malignancy of histological or pathologically confirmed diagnosis:

Omicron unresectable or metastatic melanoma (study cohort 1A)

Advanced, recurrent or metastatic squamous cell carcinoma of the head and neck (study cohort 2A)

Group III or IV NSCLC (squamous, non-squamous, adenocarcinoma, large cell carcinoma) (study cohorts 3A and 3B).

2) Only study queues 1A, 2A and 3A: the patient did not receive immunotherapy. If previously treated, the patient progresses at the time of the last treatment or after the treatment. Study queues 1A, 2A and 3A received up to 3 previous systemic anticancer therapies, specifically:

o in study cohort 1A: patients have unresectable or metastatic melanoma (stage IIIC or IV); if the BRAF mutation is positive, the patient may receive a BRAF inhibitor.

O in study cohort 2A: patients have unresectable or metastatic HNSCC. Those that have received the initial chemotherapy are allowed.

Omicron in study cohort 3A: patients had stage III or IV NSCLC (squamous, non-squamous, adenocarcinoma, or large cell carcinoma) and had not received immunotherapy in locally advanced or metastatic settings and progressed after ∈3 lines of previous systemic therapy. Patients receiving systemic therapy or receiving systemic therapy as part of definitive chemotherapy in a supplementary or neoadjuvant setting are eligible to participate and are considered to have received an on-line therapy if the disease progresses within 12 months of completing the previous systemic therapy. Patients with known oncogene drivers (e.g., EGFR, ALK, ROS) and with mutations sensitive to target therapies must progress after at least 1 online target therapies.

3) Study queue 3B only: patients with stage III or IV NSCLC (squamous, non-squamous, adenocarcinoma, or large cell carcinoma) who previously received CPI systemic therapy (e.g., anti-PD-1/anti-PD-L1) as part of.ltoreq.3 previous online systemic therapies.

Patients were subject to radiation-confirmed progress at or after the last treatment.

Patients receiving systemic therapy or receiving systemic therapy as part of definitive chemotherapy in a helper or neoadjuvant setting were eligible to participate, and were considered to have received 1 on-line therapy if the disease progressed within 12 months of completing the previous systemic therapy.

Patients with known oncogene drivers (e.g., EGFR, ALK, ROS) and with mutations sensitive to target therapies must progress after at least 1 on-line target therapies.

4) Patients have at least 1 resectable lesion (or aggregate lesion) with a minimum diameter of 1.5cm after resection for TIL research product production. Tumor tissue is encouraged to be obtained from multiple and diverse metastatic lesions, provided that surgical resection does not pose additional risk to the patient.

If a lesion for TIL production that is considered resected is located within the previously irradiated range, the lesion must show radiological progression prior to resecting.

The omicronpatient must have the appropriate histopathological samples for the planned tests.

5) Patients had residual measurable disease after tumor resection for TIL manufacture, as defined by standard RECIST 1.1 guidelines (see, e.g., eisenhauer, eur j. Cancer 2009,45,228-247):

the foci in the previously irradiated area are not selected as target foci unless the foci have shown disease progression;

the omicron can select lesions that were partially resected for TIL production and still measurable as RECIST as non-target lesions, but not as target lesions for response assessment.

6) When subject consent is signed, the patient is greater than or equal to 18 years old.

7) The patient had an eastern cancer clinical research Cooperation organization (ECOG) physical stamina of 0 or 1 and estimated life expectancy of > 3 months.

8) Patients with fertility or fertility of a companion must be willing to perform an approved highly effective method of birth control for 12 months during treatment and after receiving all of the schedule-related therapies (note: women with reproductive ability should use effective contraception during treatment and 12 months after their last dose of IL-2 or 4 months after their last dose of pembrolizumab (the latter occurrence). Men cannot donate sperm during the study or 12 months after discontinuation of treatment (later, the latter occurrence).

9) The patient had the following hematological parameters:

the number of the Absolute Neutrophils (ANC) of the omicron is more than or equal to 1000/mm ³ ；

The o hemoglobin is more than or equal to 9.0g/dL;

the number of the blood platelets of the omicron is more than or equal to 100,000/mm ³ 。

10 Patient has appropriate organ function):

serum alanine Aminotransferase (ALT)/serum glutamate pyruvate aminotransferase (SGPT) and aspartate Aminotransferase (AST)/SGOT is less than or equal to 3 times the Upper Limit of Normal (ULN), and liver metastasis patient is less than or equal to 5 times ULN.

The estimated creatinine clearance at omicron screening was ≡40mL/min using the Cockcroft Gault formula.

Total bilirubin is less than or equal to 2mg/dL.

Patients with Gilbert syndrome must have total bilirubin < 3mg/dL.

11 Patients were seronegative for human immunodeficiency virus (HIV 1 and HIV 2). Serological patient compliance positive for hepatitis B virus surface antigen (HBsAg), hepatitis B core antibody (anti-HBc) or hepatitis C virus (anti-HCV) that indicates acute or chronic infection depends on the viral load based on Polymerase Chain Reaction (PCR) and the local prevalence of certain viral exposures.

12 Patient had a minimum period of withdrawal for the previous anti-cancer therapy prior to the first study treatment (i.e., NMA-LD or pembrolizumab initiation), as detailed below:

o target therapy: previous target therapies for Epidermal Growth Factor Receptor (EGFR), MEK, BRAF, ALK, ROS1, or other target agents (e.g., erlotinib, afatinib, dacomitinib, axitinib, crizotinib, ceritinib, or loratinib (loretinib)) are allowed, provided there is a minimum 14 day withdrawal period before starting treatment.

O chemotherapy: adjuvant, neoadjuvant or definitive chemotherapy/chemotherapy is allowed as long as there is a minimum 21 days withdrawal period before starting treatment.

Study group 3B immunotherapy alone: previous checkpoint target therapies, other mabs or vaccines against PD-1 were allowed as long as there was a drug withdrawal period of ≡21 days before NMA-LD was started.

Palliative radiotherapy: the prior external beam radiation is allowed as long as all radiation-related toxicities are alleviated to grade 1 or baseline, but excluding the onset, skin pigmentation changes, or other clinically insignificant events, such as small area radiodermatitis or a feeling of urgency or urgency.

The tumor lesions assessed as response targets for RECIST 1.1 were located outside the radial port; however, if inside the radiation portal, the lesion must show progression (see inclusion criteria above).

Omicron surgery/preplanned procedure: the previous surgical procedure is allowed as long as the wound has healed, all complications have been resolved, and at least 14 days have passed before the tumor was resected (significant surgical procedure).

13 Prior to study cohort assignment, patients had recovered from all prior anti-cancer treatment-related adverse events (TRAE) to a grade of ∈1 (according to the common term criteria for adverse events [ CTCAE ]), except for the onset or leukoplakia.

14 Patients with stable, grade No. 2 toxicity due to previous anti-cancer therapies were considered case by case after medical monitors.

15 Study cohorts 1A, 2A, and 3A patients with irreversible toxicity that would be unreasonably expected to be worsened by pembrolizumab treatment were included only after negotiating medical monitors. For patients in study cohort 3B alone, patients with diarrhea or colitis noted to be rated > 2 or above 2 due to previous immune checkpoint inhibitor CPI treatment must have been asymptomatic for at least 6 months prior to tumor resection or have normal post-treatment visual assessment of colonoscopy.

16 A patient must provide written use of the protected health information and public authorization.

Exclusion criteria. Patients meeting any of the following criteria will be excluded from the study:

1) Patients with uveal/eye-derived melanoma.

2) Patients who received organ allografts or prior cell transfer therapies over the past 20 years, including non-myeloablative or myeloablative chemotherapy regimens (note: this criterion applies to patients undergoing TIL retreatment, with the exception that their previous TIL treatment had a previous NMA-LD regimen).

3) Patients with symptomatic and/or untreated brain metastases.

Patients with definitive treated brain metastases will consider a case taking after discussion with medical monitors; the conditions were that the patient had clinical stability ∈2 weeks or more before starting treatment, that Magnetic Resonance Imaging (MRI) had no new brain lesions after treatment and that the patient did not require continuous corticosteroid treatment.

4) Patients receiving systemic steroid therapy were received within 21 days of the regimen.

5) Pregnant or lactating patients.

6) Patients with active medical conditions that researchers consider to be responsible for increased risk of study participation; such as systemic infections (e.g. syphilis or any other infection requiring antibiotics), coagulation disorders or other active significant medical diseases of the cardiovascular, respiratory or immune system.

7) Patients must not have active or previously described autoimmune or inflammatory disorders (including pneumonia, inflammatory bowel disease (e.g., colitis or Crohn's disease), diverticulitis (with the exception of diverticulosis), systemic lupus erythematosus, sarcoidosis or Wegener's syndrome (granulomatous polyangiitis, graves ' disease, rheumatoid arthritis, pituitary, uveitis, etc.). The following exceptions to this standard:

Patients with o leukoplakia or hair.

Patients with hypothyroidism stabilized by hormone supplementation (e.g., after Hashimoto syndrome).

Any chronic skin condition where systemic therapy is not required.

The celiac disease patient who only needs to be drink and eat control.

8) Patients receiving either live or attenuated immunizations were received 28 days prior to initiation of treatment.

9) Patients with any form of primary immunodeficiency (e.g., severe combined immunodeficiency syndrome [ SCID ] and acquired immunodeficiency syndrome [ AIDS ]).

10 A patient who has a history of allergies to any of the components of the study medication. TIL is not administered to patients known to be allergic to any component of the TIL product formulation, including but not limited to any of the following:

o NMA-LD (cyclophosphamide, mesna and fludarabine)

οAldi interleukin, IL-2

O aminoglycoside antibiotics (i.e. streptomycin, gentamicin [ those negative for gentamicin allergy skin test excluded) ]

Any component of the o TIL product formulation, including dimethylsulfoxide [ DMSO ], HSA, IL-2, and dextran-40

Omicron-shaped pembrolizumab

11 Left ventricular ejection rate (LVEF) <45% or new york heart association class II or higher. The cardiac stress test shows any irreversible wall movement abnormality in any patient aged 60 or older or having a history of ischemic heart disease, chest pain or clinically significant atrial and/or ventricular arrhythmias.

Patients with abnormal cardiac stress tests can be documented if they have the appropriate rate of ejection and cardiac exam approved by the medical monitor of the test principal (cardiology clearance).

12 A patient with obstructive or restrictive lung disease and the noted FEV1 (forced expiratory volume at 1 second). Ltoreq.60% of the expected normal value.

If the patient cannot make a reliable spirometry due to abnormal upper airway anatomy (i.e. tracheostomy), the lung function is assessed using a 6-minute walk test. Patients who were unable to walk at least 80% of the age and sexually-expected distance or displayed evidence of hypoxia (SpO 2< 90%) at any time point during the test were excluded.

13 Patients with another primary malignancy within the previous 3 years (except those that do not require treatment or have been curative treatment more than 1 year ago and do not pose significant recurrent risk as judged by the investigator, including but not limited to non-melanoma skin cancer, DCIS, LCIS, prostate cancer gleason score +.6 or bladder cancer).

14 For 21 days of initiation of treatment, with another study product.

Study endpoint and planning analysis. The primary and secondary endpoints of the study cohort were analyzed separately.

Primary endpoint: ORR is defined as the proportion of patients who achieved a confirmed PR or CR as the best response in the efficacy analysis set as assessed by the investigator in RECIST 1.1.

Objective responses were assessed as per disease assessment, with ORR expressed as a two-term probability with a corresponding bilateral 90% CI. When all treated patients for each study cohort had the opportunity to track for 12 months, the primary analysis of each study cohort occurred unless the progress/expiration or evaluation period was prematurely aborted.

The primary endpoint of safety was expressed as two probabilities with corresponding bilateral 90% CI as measured by any TEAE incidence of grade 3 or higher within each study cohort.

Secondary endpoint: the secondary efficacy endpoint was defined as follows:

the CR ratio is based on the responders achieving the confirmed CR assessed by the investigator. DCR is the sum of the number of patients derived to achieve either confirmed PR/CR or sustained SD (at least 6 weeks) divided by the number of patients in the efficacy analysis set x 100%. CR ratio and DCR use point estimation and bilateral 90% CI summary.

DOR is defined between patients who achieve objective responses. It is measured from the date of first meeting first response (PR/CR) criteria until the first objective record of recurrent or progressive disease or receipt of subsequent anti-cancer therapy or patient death (based on the first recorded). The event time for patients who did not experience PD or did not die prior to data interception or final database locking will be limited to the last date that appropriate assessment of tumor status was made.

PFS is defined as the time (months) from lymphocyte depletion to PD or death for any reason (based on the event that occurred earlier). The event time of patients who did not experience PD or did not die at the time of data interception or final database locking is limited to the last date that appropriate assessment of tumor status was made.

OS is defined as the time (months) from lymphocyte depletion to death for any reason. The event time for patients that have not died prior to the data interception or final database locking is limited to the last date of their known survival status.

DOR, PFS and OS are right bounded. The Kaplan-Meier method will be used to outline the end point of the time to onset of the event. Baseline data for tumor assessment was the last scan prior to lymphocyte depletion for all study cohorts.

The efficacy parameters described above will be estimated for the subset of applicable study cohorts defined by baseline disease characteristics; BRAF status (study cohort 1A only), HPV status (study cohort 2A only), squamous or non-squamous pulmonary disease (study cohorts 3A and 3B only), and anti-PD-L1 status.

Example 15: stage 2, multicenter study of autologous tumor-infiltrating lymphocytes in patients with locally advanced or metastatic non-small cell lung cancer

This example relates to the treatment of patients with locally advanced unresectable or metastatic non-small cell lung cancer (NSCLC) without any actionable driving mutations, who underwent disease progression upon or after approved systemic therapy on a single line consisting of the combination checkpoint inhibitor (CPI) +chemotherapy ± bevacizumab (including bevacizumab, VEGFA inhibitors), the study cohort of treatments is summarized below:

study cohort 1: patients whose tumors did not express programmed cell death ligand 1 (PD-L1) (tumor fraction [ TPS ] < 1%) prior to their CPI treatment.

Study cohort 2: patients whose tumors expressed PD-L1 (TPS. Gtoreq.1%) prior to their CPI treatment. Study cohort 3: patients whose tumors did not express PD-L1 (TPS < 1%) prior to their CPI treatment and were not able to safely perform surgical collection for TIL production because at least one of the following:

the risk of unacceptable surgery, or

The omicrons require surgically accessible lesions for solid tumor response assessment criteria (RECIST) v1.1 assessment.

Study cohort 4: retreatment study cohort: patients previously treated with TIL-based immunotherapy in study cohorts 1, 2 or 3 of the present study.

Treatment will be administered using autologous TIL-based immunotherapy derived from the tumor of the individual patient for patient-guided therapy.

The TIL-based immunotherapy treatment regimen involves a NMA-LD preparation regimen course of 5 days total using cyclophosphamide and fludarabine prior to the TIL-based immunotherapy infusion, and a limited course of IL-2 administration (up to six doses) following the TIL-based immunotherapy infusion. NMA-LD preparation protocol and IL-2 are included in the protocol to support implantation, amplification and activation of transferred TILs.

Several preparatory regimens have been used in conjunction with TIL therapy. NMA-LD preparation protocols include cyclophosphamide/fludarabine, total Body Irradiation (TBI), or a combination of both. This exemplary study utilized the cy-flu protocol. The NMA-LD preparation protocol currently used for research is a method based on the National Cancer Institute (NCI) development and testing that involves 2 days cyclophosphamide and 5 days fludarabine to shorten patient hospitalization days. Each patient will undergo an NMA-LD preparation regimen prior to TIL-based immunotherapy infusion.

The therapy is an autologous TIL-based immunotherapy ready for infusion. TIL-based immunotherapy consists of autologous TIL, which is obtained from tumors of individual patients and expanded ex vivo by cell culture in the presence of the cytokines IL-2 and a murine monoclonal antibody (mAb) against human CD3 (OKT 3).

The final drug product was a cryopreserved viable cell suspension formulated for IV infusion. The ex vivo amplified autologous TIL is formulated inCS10 cryopreservation Medium/Plasmalyte (final dimethyl sulfoxide [ DMSO ]]Concentration: 5%) and contains 0.5% Human Serum Albumin (HSA) and 300IU/mL (12 ng/mL) IL-2. The formulated product is frozen at a controlled rate in gaseous liquid nitrogen to<-150 ℃, transported in frozen shipping tanks to the appropriate clinical center, thawed prior to infusion into the patient.

The manufacturing process begins with surgical excision at a clinical center or core biopsy containing a tumor lesion of viable tumor material. Aggregates of multiple separate focal biopsy sections may also be encouraged to be excised from the patient, as allowed by patient safety. Tumor samples were placed in transport medium and delivered by express delivery to Good Manufacturing Practice (GMP) manufacturing facility at 2 to 8 ℃. Upon reaching the GMP manufacturing facility, the tumor sample is fragmented and then activated (initial amplification step) to produce the minimum number of viable cells required for the rapid amplification protocol (REP) phase. Tumors may also be enzymatically dissociated and biomarker expression of TIL may be selected prior to REP. The REP stage (second expansion step) further expands cells in the presence of IL-2, OKT3 and irradiated allogeneic Peripheral Blood Mononuclear Cells (PBMCs). The REP expanded cells are then collected, washed and formulated in a blood transport/infusion bag for delivery to a clinical center by express delivery. The manufacturing process of TIL-based immunotherapy is illustrated as provided in fig. 34 and 35.

Each cryopreservation bag of TIL-based immunotherapy end-product is labeled with a patient-specific label. TIL-based immunotherapy is transported from manufacturing facilities to a clinical center for administration to patients.

This example pertains to the evaluation of TIL-based immunotherapy for prospective, open label, multiple study cohorts, non-randomized group, multicenter phase 2 study in locally advanced unresectable or metastatic NSCLC patients.

The following study cohorts were studied:

study cohort 1: TIL-based immunotherapy is used as a monotherapy in stage IV NSCLC patients whose tumors do not express PD-L1 (tumor fraction [ TPS ] < 1%) prior to their CPI treatment and do not have known actionable driving mutations, who either upon or after receiving approved systemic therapy on a single line consisting of the combination cpi+chemotherapy ± bevacizumab, have at least one resectable lesion (or aggregate lesion) with a minimum diameter of 1.5cm for TIL production and at least one remaining measurable lesion as defined by RECIST 1.1 after resection.

Study cohort 2: TIL-based immunotherapy is used as a monotherapy in stage IV NSCLC patients whose tumors express PD-L1 (TPS > 1%) prior to their CPI treatment and do not have any known actionable driving mutations, who either upon or after receiving approved systemic therapy on a single line consisting of the combination cpi+chemotherapy ± bevacizumab, have at least one resectable lesion (or aggregate lesion) with a minimum diameter of 1.5cm for TIL production and at least one remaining measurable lesion as defined by RECIST 1.1 after resection.

Study cohort 3: TIL-based immunotherapy is used as a monotherapy in stage IV NSCLC patients whose tumors do not express PD-L1 (TPS < 1%) prior to their CPI treatment and do not have any known actionable driving mutations, patients develop disease upon or after receiving approved systemic therapy on a single line consisting of the combination cpi+ chemotherapy ± bevacizumab, and patients collected for surgery for TIL production cannot be safely performed because at least one of the following: 1) Unacceptable surgical risk or 2) surgical accessibility to lesions is required for RECIST assessment.

Study cohort 4: TIL-based immunotherapy monotherapy as a retreatment of patients who previously received TIL-based immunotherapy as part of their participation in study cohorts 1, 2, or 3.

For study cohorts 1, 2, 3 and 4, all patients received autologous cryopreservation TIL-based immunotherapy and previously received a non-myeloablative lymphocyte depletion (NMA-LD) pretreatment regimen consisting of cyclophosphamide and fludarabine. Up to 6 doses of IV IL-2 (e.g., aldesleukin or a biological analog or variant thereof) are administered following TIL-based immunotherapy infusion. Alternatively, downstream IL-2 or low dose IL-2 may be used as listed herein.

Autologous TIL therapy comprising TIL-based immunotherapy comprises the following general steps:

tumor collection to provide autologous tissue that acts as a source of autologous TIL cellular products,

the production of an autologous TIL-based immunotherapeutic research product (IP) at a central Good Manufacturing Practice (GMP) agency,

5 days non-myeloablative lymphocyte depletion (NMA-LD) pretreatment protocol,

infusion of TIL-based immunotherapy products (day 0), and

administering 6 or less doses of IV IL-2.

The main objective was to evaluate the efficacy of TIL-based immunotherapy for locally advanced unresectable or metastatic NSCLC patients without actionable driving mutations, who received approved systemic therapy on a single line consisting of combined checkpoint inhibitor (CPI) +chemotherapy ± bevacizumab or later disease progression as judged by independent evaluation committee (IRC) (study cohorts 1 and 2) or by researchers (study cohorts 3 and 4) using the solid tumor response evaluation criteria (RECIST 1.1) to evaluate Objective Response Rate (ORR).

A secondary objective was to assess the efficacy of TIL-based immunotherapy, as judged by the investigators (study cohorts 1 and 2) using RECIST 1.1 to evaluate ORR, further using Complete Response (CR) rates; duration of reaction (DOR); disease Control Rate (DCR); progression Free Survival (PFS) assessed the efficacy of TIL-based immunotherapy as assessed by IRC (study cohorts 1 and 2) and researchers (all study cohorts) using RECIST 1.1; and Overall Survival (OS), and characterize the safety profile of TIL-based immunotherapy in NSCLC patients, as measured by ≡3-level treatment-induced adverse event (TEAE) incidence. For study-only cohort 3, the efficiency of producing TIL-based immunotherapy from core biopsy was assessed.

Purpose of exploratory use: (1) The persistence and recognition of TIL-based immunotherapy can affect immune-related factors of response, outcome and toxicity variables. (2) Individual indication specific health related quality of life (HRQoL) parameters were assessed.

Primary endpoint: ORR was assessed by IRC (study cohorts 1 and 2) or by the investigator (study cohorts 3 and 4) as RECIST 1.1.

Secondary endpoint: (1) The incidence and characteristics of treatment-induced adverse events (TEAEs) in relation to the severity of the study treatment, the severity of the serious nature, including Severe AE (SAE), therapy-related AEs, and AEs that lead to early discontinuation of treatment or withdrawal from the evaluation period or death. (2) CR (complete response) rate, DOR (duration of response), DCR (disease control rate) and PFS (progression free survival), as assessed by IRC in accordance with RECIST 1.1 (study cohorts 1 and 2). (3) ORR (objective response rate), CR rate, DOR, DCR and PFS, as assessed by the investigator in accordance with RECIST 1.1 (all study cohorts). (4) OS (overall survival). (5) The percentage of successful TIL product produced by core biopsy (study cohort 3).

Exploratory endpoint: the in vivo persistence of T cells (containing TIL product) was assessed by monitoring the presence of TIL product specific T cell receptor beta complementarity determining region 3 (CDR 3) sequences in the patient's blood over time. Identification of blood samples present in the product and periphery using deep sequencing CDR3 sequences of (b).

Also included are exploratory endpoints aimed at identifying predictive and pharmacodynamic clinical biomarkers of activity of TIL-based immunotherapy:

phenotypic and functional characteristics of TIL-based immunotherapy;

immune profile of tumor tissue;

gene expression profile of TIL products, tumor tissue and/or PBMCs;

mutant appearance of tumor;

circulating immune factors; and

immune composition of PBMC.

HRQoL (health related quality of life) assessed according to european cancer research and therapy organisation (EORTC) Quality of Life Questionnaire (QLQ) C30 and QLQ LC13 is also included.

Study design details: TIL-based immunotherapy Adoptive Cell Therapy (ACT) was evaluated for prospective, open label, multiple study cohorts, non-randomized group, multiple center phase 2 study.

All patients received TIL-based immunotherapy consisting of these steps:

tumor collection provides autologous tissue that acts as a source of autologous TIL cellular products,

in the production of an autologous TIL-based immunotherapeutic research product (IP) at a central agency operating according to Good Manufacturing Practice (GMP),

5 days non-myeloablative lymphocyte depletion (NMA-LD) pretreatment protocol,

infusion of TIL-based immunotherapy products (day 0), and

Administering 6 or less doses of IV IL-2.

The following regular sequential periods will occur in all 4 study queues unless otherwise indicated:

1. screening period: self-Informed Consent (ICF) signature to receipt

2. Pretreatment period: from case collection to initial preparation of NMA-LD protocol.

3. Treatment period: back diagnosis from initial preparation NMA-LD to end of treatment (EOT). This consisted of 8 to 9 days of therapy, including NMA-LD (day-5 to-1), TIL-based immunotherapy infusion (day 0), followed by IL-2 administration (day 0 or 1 to 3 or 4). EOT occurs about 30 days after day 0.

4. A post-treatment follow-up period consisting of:

a. post-treatment efficacy follow-up (TEFU): from EOT back diagnosis to study completion (5 years after treatment [ month 60 ]) or end of efficacy assessment (EOEA) back diagnosis will be suggested by disease progression or start of new anti-cancer therapy, whichever occurs first.

b. Long-term tracking period (LTFU): from EOEA as described above to completion of the study (5 years after treatment [ month 60 ]).

Study participants (docket patients) will switch to LTFU early (e.g., partially withdraw consent, or if for any reason decide not to receive TIL-based immunotherapy). Early study withdrawal was prompted by withdrawal of consent, death, loss of follow-up, or termination of the study by the trial delegate. A flow chart of the study design is presented in fig. 36.

Patients will undergo a 5 day pretreatment NMA-LD regimen that is initiated prior to the scheduled day 0 (i.e., -5 to-1) TIL-based immunotherapy infusion. NMA LD regimen consists of 2-day Intravenous (IV) cyclophosphamide (60 mg/kg) plus mesna (mesna) (care or USPI/SmPC according to the center standard) and 5-day fludarabine IV (25 mg/m) on day-5 and day-4 ² Day-5 to day-1).

IL-2IV was administered at a dose of 600,000IU/kg, beginning as soon as 3 hours but no later than 24 hours after completion of the TIL-based immunotherapy infusion on day 0. Additional doses of IL-2 were administered about once every 8 to 12 hours, up to a total of 6 doses.

Table 83: therapeutic administration regimen

^a () If applicable

Preparation of mesna: mesna was administered to reduce the risk of hemorrhagic cystitis associated with cyclophosphamide administration. Mesna is administered as continuous or intermittent infusion according to local standards.

If the amount of cyclophosphamide is reduced, the total dose of mesna is not adjusted. The volume of mesna injection or infusion was diluted according to institutional standards.

Infusion of cyclophosphamide and mesna: cyclophosphamide (60 mg/kg) in a total volume of 250mL or 500mL (e.g., 5% dextrose in water [ D5W)]Or 0.9% sodium chloride [ NaCl ]]) Is a kind of medium. Mesna (15 mg/kg) was infused with cyclophosphamide over about 2 hours (on days-5 and-4) via continuous infusion followed by infusion in a suitable diluent at a rate of 3 mg/kg/hour for the remaining 22 hours within 24 hours beginning simultaneously with each cyclophosphamide dose. The total dose administered is at least 1.3 times the dose of cyclophosphamide. Higher or sustained doses of mesna may be administered to prevent hemorrhagic cystitis.

Infusion of fludarabine: fludarabine (25 mg/m) ² ) IV administration is once daily for about 30 minutes for 5 consecutive days during the-5 day to-1 day period.

Participation period: overall, the study participation time will be from self-treatment to completion for up to 5 years.

Selecting inclusion criteria：

NSCLC with histological or pathological diagnosis (squamous, non-squamous, adenocarcinoma, large cell or mixed histological), the PD-L1 expression status (i.e. historical TPS of informed initial treatment selection) as judged by tumor fraction (TPS) before it receives CPI treatment must have been documented (TPS <1% for study cohorts 1 and 3, TPS > 1% for study cohort 2).

Systemic therapy on a single line has been received, including simultaneous CPI and chemotherapy, with radiological disease progression noted at or after the receipt of systemic therapy on this single line.

If the disease does not progress during or within 12 months of completion of the therapy, previous systemic therapies in a helper or neoadjuvant setting or as part of definitive chemotherapy do not account for the on-line therapy. Previous TIL treatments at this protocol did not account for the on-line therapy of study cohort 4 (re-treatment) patients.

Exercise tolerance has been described as not less than 85% of its expected normal range of age and without signs or symptoms of ischemia or clinically significant arrhythmia.

Have an eastern cancer clinical research co-ordination organization (ECOG) physical stamina of 0 or 1 in the united states and the researchers consider estimated life expectancy >6 months.

Study queues 1 and 2: it is necessary to have at least one resectable lesion (or aggregate lesion) with a minimum diameter of 1.5cm for TIL production.

Study queue 3 only: the patient must have a single RECIST 1.1 measurable lesion and no additional lesions available for surgical collection, or cannot safely perform surgical collection for TIL production, but can safely perform sufficient tumor collection for TIL production by radiologically guided core biopsy.

Study cohort 4: following either paradigm.

All study queues: if a lesion for collection is considered to be within the previously irradiated range, the lesion must show radiological progress prior to collection and irradiation must be completed at least 3 months prior to filing. The patient must have the appropriate histopathological sample for the planned test.

After tumor collection for TIL manufacture, all patients must have at least one measurable lesion remaining as defined by RECIST 1.1, with the following considerations:

lesions in previously irradiated areas were not selected as target lesions unless the lesions had shown progression and irradiation had been completed at least 3 months prior to receipt.

Study queues 1 and 2 only: lesions that were surgically partially resected for TIL production and remain measurable according to RECIST v1.1 may be selected as non-target lesions, but not as target lesions for response assessment.

Study queue 3 only: if no other lesions are available for core biopsy generated by TIL, a single RECIST v1.1 measurable lesion may serve as both a core biopsy collection site and a lesion to monitor the response.

Study cohort 4: either paradigm can be followed, but must have at least one focus of RECIST v1.1 measurable to track the response.

The following efficacy parameters of TIL-based immunotherapy as monotherapy for NSCLC patients were investigated in each study cohort: ORR, CR, DOR, DCR, PFS and OS.

Statistical analysis is based on estimates of efficacy and safety parameters and will be performed on a study cohort. No formal statistical comparisons were applied between study cohorts. The primary efficacy endpoint was the ORR assessed by IRC (study cohorts 1 and 2) or by the investigator (study cohorts 3 and 4) according to RECIST v 1.1. ORR, CR Rate and DCR were based on Clopper-Pearson accurate methods using point estimation and double sided 95% trust limit overview. The Kaplan-Meier method is used to outline the time-to-effect endpoint required for event occurrence, such as DOR, PFS and OS. DOR analysis was performed on patients who achieved objective responses. Safety analysis is descriptive and is based on an overview of TEAE, SAE and AE, vital signs and clinical laboratory tests that lead to study discontinuation.

The total number of planned patients infused with TIL-based immunotherapy in study cohorts 1, 2 and 3 was about 95. For study cohorts 1 and 2, about 40 patients were selected for each study cohort. For each study cohort, a Simon two-stage design (Simon, 1989) with a large-medium-small (minimum) was used to test the null (null) hypothesis of less than or equal to 10% ORR versus the surrogate hypothesis of >10% ORR. Twenty-five patients should be counted in the first stage. If 2 or less of these 25 patients respond to therapy, the study cohort may be terminated. Otherwise, a total of 40 patients amplified to stage 2 occurred simultaneously with the analysis of stage 1. At the end of the second phase, if at least 7 out of a total of 40 patients respond to the therapy, the null hypothesis is rejected. This 2-phase design provides 70% assay force rejecting 10% ORR null hypothesis based on assuming a single-sided alpha level of 0.1 for TIL-based immunotherapy 20% ORR. For study cohort 3, approximately 15 patients were planned, which provided estimated ORR by the Clopper-Pearson exact method, 90% Confidence Interval (CI) half width <0.23. In the case of study cohort 4 (retreatment study cohort), patients previously treated with TIL-based immunotherapy in study cohorts 1, 2 or 3 of the present study were enrolled.

Example 16: identifying tumor markers suitable for chimeric costimulatory receptor design

This example describes the identification of tumor markers suitable for CCR and TIL modified to express CCR. Flow cytometry analysis of tumor samples was used to measure the expression of two markers EPCAM and TROP-2 described elsewhere herein. Using the BD Canto II system, flow cytometry was performed using antibodies to EPCAM, EPCAM-PE (BD, catalog number 566841) and APC (BD, catalog number 566842), clone 9C4, and antibodies to TROP-2, TROP-2PE (BD, catalog number 564837), clone 162-46. FIG. 43 shows flow cytometry analysis of cervical cancer tumor (accession number 1911271423) digests, showing highly expressed EPCAM, TROP-2 and combinations of both markers. FIG. 44 similarly shows flow cytometry analysis of cervical cancer tumor digests using EPCAM-APC instead of EPCAM-PE. FIG. 45 shows flow cytometry analysis of EPCAM/TROP-2 expression on head and neck squamous cell carcinoma tumor (accession number H3103) digests. FIG. 46 shows EPCAM/TROP-2 expression on non-small cell lung cancer tumor (accession number L4172) digests. Higher EPCAM and TROP-2 expression were consistently observed in multiple tumor types and replicas, showing applicability with CCR targeting these molecules for the cancers described herein.

Example 17: preparation of chimeric costimulatory receptor modified TIL products

Lentiviral vectors may be prepared to produce CCRs comprising an extracellular PD-1 binding domain (e.g., as set forth in SEQ ID NO:244, SEQ ID NO:245 or SEQ ID NO: 246). Three versions of this CCR can be prepared using the domains shown in FIG. 36, namely the complete signaling domain of CD28 (YNM+PRRP+PYAP motif), the partial signaling domain (YNM+PRRP motif) and the YNM signaling domain alone (see SEQ ID NO: 572).

Cloning methods as described further herein and/or as known in the art may also be used to construct a nucleic acid comprising SEQ ID NO: 618. SEQ ID NO:619 or SEQ ID NO:620 corresponds to the lentiviral vector of FIG. 38, FIG. 39 or FIG. 40, respectively.

For packaging, the lentiviral vector described above can be co-transfected with VSV-G envelope plasmid and Gag/Pol and Rev packaging plasmids into HEK293T packaging cells. After about two days of incubation, the supernatant was collected, centrifuged to remove the residue and filtered. Lentiviral particlesThe seed was concentrated using polyethylene glycol and further purified by sucrose gradient buffered ultracentrifugation. Green fluorescent protein transgenes can be added to assess transfection efficiency of study batches prior to pilot and clinical batches. At least 1X 10 ⁹ TU/mL Transfection Units (TU) are required. Endotoxin levels and other release parameters of lentiviral products can be determined and subsequently used to transfect TIL, MILs or PBL. For example, the 2 nd generation procedure may be modified as described elsewhere herein (e.g., fig. 2A, 2B, and 2C prior to 11 th day REP initiation) to transfect TIL between the pre-REP and the REP stage, optionally with a short rest period before, during, and/or after transfection, wherein the total duration of the pre-REP stage is from about 3 to about 14 days, the total duration of the REP stage is from about 3 to about 14 days, obtaining more than 10 ⁸ Individual cells express CCR and are useful in the treatment of a therapeutic TIL population of cancers in patients from which the tumor is obtained.

The foregoing process may also be performed using retroviral and transposase expression systems as described elsewhere herein or as known in the art.

Example 18: chemokine receptor expression on TIL

To assess chemokine receptor expression on TIL, nine different TIL cell lines produced by the 2 nd generation process were thawed and stained on different days for characterization. PBMCs were used as controls. The TIL batches used and the results are summarized in table 84.

Table 84: assessment of chemokine receptor expression on 9 TIL batches prepared from different tumors (ov=ovary; ep=breast; l=lung)

Sample #)	TIL batch	Position of	Viable cell count	Viability%	Total cell #)	Volume of 1mL
							1	OV8175	T1B2R8P7	1.19×10 ⁷	90.3	11900000	84.03
2	OV8178	T1B3R3P2	8.18×10 ⁶	90.5	8180000	122.25
							3	OV8185	T1B6R4P2	1.08×10 ⁷	88.1	10800000	92.59
4	EP11143	T1B5R7P3	7.41×10 ⁶	82.6	7410000	134.95
							5	EP11145	T1B7R1P8	9.97×10 ⁶	87.6	9970000	100.30
6	EP11147	T4B3R7P6	9.47×10 ⁶	83.5	9470000	105.60
							7	L4231	T1B3R6P8	9.54×10 ⁶	67.7	9540000	104.82
8	L4233	T1B4R6P9	7.58×10 ⁶	78.7	1.52×10 ⁷	131.93
							9	L4240	T1B9R5P2	5.68×10 ⁶	81.9	5680000	176.06
10	PBMC	T7B5R3P2	8.39×10 ⁶	93.3	16780000	119.19

For flow cytometry analysis, a Bio-Rad ZE5 cell analyzer (Heracles, calif. USA) was used. For surface staining of chemokine receptors, 1X 10 ⁶ Individual cells were seeded in 96-well V-bottom plates. Cells were then washed and stained with a live/dead fixable blue dead cell staining kit of Life Technologies (carlsbad, california, usa) in the presence of a human trustin FcX Fc receptor blocking solution of BioLegend (san diego, california, usa) for 10 minutes at room temperature. Antibodies were then added to the cells and the samples incubated at 4 ℃ for 25 minutes. The samples were then washed twice, filtered and run on a ZE5 analyzer. Analysis was performed using FlowJo software. All gating was based on Fluorescence Minus One (FMO) control. The results are shown in FIGS. 47, 48, 49 and 50. A moderate level of CXCR3 was observed, which bound to CXCL9/10/11. CXCR4 and CXCR5, which bind to CXCL12 and CXCL13, respectively, can be used to locate TIL close to B cells and tertiary lymphoid structures. CXCR1 and CXCR2 bind to other ligands such as CXCL8 and can be used for movement of complex-closing (co-opt) neutrophils. CCR2 binds MCP-1 and Other ligands and can be used for movement of the close-packed monocytes. CCR4 and CCR8 bind CCL17, CCL22 (CCR 4) and CCL1 (CCR 8) and are available for movement of the aggregate tregs into tumors.

Example 19: preparation of chemokine receptor modified TIL products

VSV-G pseudotyped MSCV retroviral vectors can be prepared to produce chemokine receptors comprising CXCR1 or CCR8 domains (e.g., those shown in SEQ ID NO:627 and SEQ ID NO:632, respectively). For packaging, the above vectors can be co-transfected with VSV-G envelope plasmids and Gag/Pol and Rev packaging plasmids into HEK293T packaging cells. After about two days of incubation, the supernatant was collected, centrifuged to remove the residue and filtered. The virions were concentrated using polyethylene glycol and further purified by sucrose gradient buffered ultracentrifugation. Green fluorescent protein transgenes can be added to assess transfection efficiency of study batches prior to pilot and clinical batches. At least 1X 10 ⁷ TU force valences of TU/mL are desired. Endotoxin levels and other release parameters of the retrovirus products can be determined for subsequent use in transfection of TIL, MIL or PBL. For example, the 2 nd generation procedure may be modified as described elsewhere herein (e.g., fig. 2A, 2B, and 2C prior to 11 th day REP initiation) to transfect TIL between the pre-REP and the REP stage, optionally with a short rest period before, during, and/or after transfection, wherein the total duration of the pre-REP stage is from about 3 to about 14 days, the total duration of the REP stage is from about 3 to about 14 days, obtaining more than 10 ⁸ Individual cells express more than one chemokine receptor based transgene and are useful in treating a therapeutic TIL population of tumors in a cancer of a patient from which the tumor is obtained.

Exemplary CCR constructs can be used with TIL produced by the above experiments. Sequences of constructs labeled "CCR1" to "CCR6" (fig. 51) were synthesized and inserted into the pqxix vector by insertion into MCS I (fig. 52), eGFP was inserted into MCS II as an expression report. The commercially available pqxix vector is a self-inactivating lentiviral vector with gene expression driven by the CMV promoter. To prepare the retrovirus, the pqxix vector was transfected into PT67 packaging cells. Viral supernatants were collected from day 2 to day 3 culture supernatants to transduce HEK reporter cells. HEK reporter cells were transduced with retroviruses with CCR constructs. Determining expression of constructs labeled "CCR4" and "CCR5" in HEK reporter cells; the surface expression of the PD-1 extracellular domain in "CCR4" (fig. 53, panel a) and "CCR5" (fig. 53, panel b) in untransduced (GFP-) and transduced (gfp+) HEK reporter cells was determined by flow cytometry, the results of which are shown in fig. 53.

Example 20: double epitope chimeric costimulatory receptor

Additional CCR can be prepared using the methods described above. FIG. 54 shows constructs of bi-epitope CCRs, each designed to include 2 CCRs in a bi-cistron construct separated by a T2A domain. The amino acid sequences of the labeled domains of these CCR are given in fig. 55 to 60 and table 85. CD 8a leader peptide was used in all constructs. For PD-L1 CCR, 19H9 and 38A1 scFv domains were selected to potentially target two epitopes of PD-L1 (SEQ ID NO:658 and SEQ ID NO: 659).

For the anti-TROP-2 CCR construct, two versions were designed. TROP-2 can potentially form dimers, although not being limited by theory, it is believed that one scFv (h 6G11, SEQ ID NO: 316) rather than two scFv may be sufficient to bring together unique intracellular subunits to form a complex and transmit downstream activation signals. Thus SEQ ID NO:660 and SEQ ID NO:661 includes this feature. However, if TROP-2 does not form dimers, two scFv strategies are included and clones cAR47A6.4 and KM4097 (SEQ ID NO:662 and SEQ ID NO: 663) are used that do not compete in the competition assay. Suitable, non-limiting embodiments of CCRs prepared according to this example and useful as CCR constructs of the invention are shown in Table 85.

Table 85: amino acid sequence of exemplary double epitope CCR

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The foregoing examples (which are also embodiments of the present invention) provide for expression of SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 has at least 99% identity to the sequence set forth in SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 has at least 98% identity to the sequence set forth in SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 has at least 97% identity to the sequence set forth in SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 has at least 96% identity to the sequence set forth in SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 has at least 95% identity to the sequence set forth in SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 has at least 90% identity to the sequence set forth in SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676, or a sequence having at least 85% identity to SEQ ID NO: 671. SEQ ID NO: 672. SEQ ID NO: 673. SEQ ID NO: 674. SEQ ID NO:675 or SEQ ID NO:676 has an amino acid sequence with at least 80% identity.

In one embodiment, these constructs consist of SEQ ID NO: 621. SEQ ID NO: 622. SEQ ID NO: 623. SEQ ID NO: 624. SEQ ID NO:625 and SEQ ID NO: 626. In one embodiment, the CCR of the present invention comprises a composition comprising the foregoing bi-epitope CCR expressed by one of the foregoing bi-cistron constructs. The eGFP domain can be removed for use in preparing TIL for human therapy.

CCR7 to CCR12 sequences were synthesized (as shown in fig. 54) and inserted into the slenti viral vector by replacing the Cas9-GFP cassette for the gene of interest (fig. 61). The pLenti virus vector is a self-inactivating lentiviral vector with gene expression driven by EF-1 alpha core promoter. To prepare lentiviruses, the pLenti vector and helper vector (VSV-G, gag/Pol) were co-transfected into 293T cells. Viral supernatants were collected from day 2 to day 3 culture supernatants to transduce HEK reporter cells.

Exemplary results showing expression of bi-epitope CCR8 and CCR12 constructs in HEK reporter cells are presented in fig. 62. HEK reporter cells were transduced with lentivirus with the CCR8 and CCR12 constructs shown. In FIG. 62, the results of HEK-IL-18 reporter cells transduced with CCR8 and incubated with biotin-conjugated PD-L1 protein are shown in panel (A), and the results of HEK-IL-18 reporter cells transduced with CCR12 and incubated with biotin-conjugated TROP-2 protein are shown in panel (B), each showing the expression of the desired CCR after fluorescent staining with streptavidin.

The competitive binding of these antibodies was then investigated to determine the feasibility of the bi-epitope CCR construct. Characterization data for anti-PD-L1 antibody clones 38A1-IgG4-HA and 19H-IgG4-Flag are given in Table 86.

Table 86: characterization of anti-PD-L1 Ab clone 38A1-IgG4-HA and 19H-IgG4-Flag

	EC50	Ab stock solution	MW
				38A1-IgG4-HA	0.4039nM	0.45μg/μL	54.162kDa
19H9-IgG4-Flag	0.1248nM	0.96μg/μL	55.481KDa

First, hPD-L1Raji cells were incubated with 38A1-IgG4-HA antibody targeting PD-L1 at the concentrations indicated (FIG. 63) in the presence of competing hPD-L1 binding antibody 19H 9. After 2 hours of incubation, cells were washed and stained for anti-HA-APC for analysis. In FIG. 63, the x-axis shows the concentration of titrated 38A1-IgG4-HA antibody and the Y-axis shows the% PD-L1 positive stained cells in total hPD-L1Raji cells. The hPD-L1Raji cells were then incubated in the presence of the competing hPD-L1 binding antibody 19H9 with the concentration of 19H9-IgG4-Flag antibody targeting PD-L1 shown (FIG. 64). After 2 hours of incubation, cells were washed and stained for anti-Flag-AF 488. In FIG. 64, the x-axis shows the concentration of titrated 19H9-IgG4-Flag antibody and the Y-axis shows the% of PD-L1 positive stained cells in total hPD-L1Raji cells. The results indicated that 38A1 and 19H9 non-competitively bound to PD-L1. FIG. 65 shows co-stained flow cytometry data for Raji cells stained with each of the antibodies shown; wherein hPDL-1-Raji cells were incubated with 19H9-IgG4-Flag Ab and 38A1-IgG4-HA Ab, followed by fluorophore conjugated secondary antibodies.

Additional experiments were performed to assess the blocking efficacy of two PD-L1 antibodies using the method depicted in fig. 66. Jurkat-Lucia ^TM TCR-hPD-1 cells (2X 10) ⁵ Individual cells) and Raji-APC-hPD-L1 cells (2X 10) ⁵ Individual cells) were co-cultured in the presence of 19H9 and 38A1 anti-PD-L1 antibodies at the indicated concentrations shown on the x-axis of figure 67. 19H9 and 38A1 are added singly or in combination to the co-cultivation system. After 24 hours, with QUANTI-Luc ^TM The assay kit quantifies the Lucia activity. The results are shown in FIG. 67.

In summary, the bi-epitope CCR construct can be successfully expressed in HEK blue reporter cells and exhibit antigen specific binding capacity. Surprisingly, PD-L1 did not competitively bind to PD-L1 clones 38A1 and 19H9, indicating that it binds to a different PD-L1 epitope. These clones were adapted for bi-epitope CCR design as shown in this example. This design can provide two subunits for the intracellular domains of IL-18R and IL-2R.

Example 21: use of AKT inhibitors to increase CD39 of TIL products ^- CD69 ^- Phenotype of phenotype

Memory progenitor cell stem cell-like (CD 39) ^- CD69 ^- ) Phenotype is associated with complete regression and TIL persistence in the study cohort of metastatic melanoma patients (Krishna et al, science 2020,370,1328). Strategies aimed at expanding TILs with lower differentiation and more stem cell-like properties may lead to improved persistence, functionality and more efficient tumor response. Pharmacological inhibition of AKT in TIL has been shown to induce transcriptional, metabolic and functional property profiles of memory T cells. In this example, AKT inhibition during ex vivo TIL expansion was studied to determine if it could increase the proportion of less differentiated, more stem cell-like cells with improved cytokine output and functionality.

Tumors from patients of different indications were received, crushed and subjected to an amplification protocol for TIL manufacture. Different doses (3 μm and 1 μm) of the pan AKT inhibitor eparatadine were added to the culture during ex vivo amplification. Amplification potential, phenotype and functional characteristics were assessed at the final TIL product. Eight tumors were used in combination in the 2 nd generation procedure, including tumors from melanoma, NSCLC, head and neck cancer, ovarian cancer, and breast cancer.

Figure 68 shows that AKT inhibitor treatment maintains TIL expansion and viability and does not affect T cell proportion. Amplification, viability and T cell distribution of control and AKT inhibitor treated TIL are shown. TIL is untreated or treated with increased concentrations of the pan AKT inhibitor eparatadine. The treatments were added before and during REP (blue bars) or during the REP-only phase (purple bars). Showing the amplification and viability of TIL at the end of the 22 day amplification process. Also shown is CD8 of cryopreserved cells after the expansion process ⁺ 、CD4 ⁺ And CD4 ⁺ (Foxp3 ⁺ ) Frequency of cells.

Additional results are shown in fig. 69-75. AKT inhibition is shown in figure 69 to induce CD8 ⁺ Increased frequency of TEMRA cells and increased CD8 is shown in figure 70 ⁺ IL-7R and CXCR3 expression on TIL. FIG. 71 shows AKT inhibition at CD8 ⁺ And CD4 ⁺ Increasing lower differentiation CD8 on both TILs ⁺ CD69 ^- CD39 ^- Frequency of T cells. FIG. 72 shows CD8 ⁺ CD69 ^- CD39 ^- TIL is less differentiated and depleted. TIL treated with AKT inhibitors also maintains higher frequency of CD8 after stimulation ⁺ CD69 ^- CD39 ^- T cells and lower TOX expression as shown in figure 73.

Cryopreserved controls and TIL treated with 1 μm eparatadine before and at both REP at 1:5 against cell scale anti-CD 3/CD28 beads were stimulated overnight. The results are shown in FIG. 74. It was observed that AKT inhibitor treated TIL prepared using the generation 2 procedure maintained higher cytokine output after stimulation.

Cryopreserved controls and TIL and 1 μm eparatadine treated both before and after REPTHP-1 cells at 10:1 effector to target cell ratio co-cultures for 24 hours to measure cytotoxicity in an allogeneic environment. TIL treated with control and eparatadine at 1 every 5 days: anti-CD 3/CD28 bead stimulation of the 1 bead versus cell scale. Three days after the third stimulation, cells were washed, beads removed, and cells were washed at 10:1 effector to target cell ratio of KILR THP-1 cells were co-cultured for 24 hours. As shown in fig. 75, epatazobactam-treated TIL prepared using the generation 2 procedure showed a sustained increase in cytotoxicity in the allogeneic environment following repeated stimulation.

Treatment with AKT inhibitor at a dose of 1 μm resulted in equivalent amplification and viability of TIL relative to control, but less differentiation of CD39 ^- CD69 ^- The cell population doubles. Even though this effect was still present after restimulation, these cells showed reduced CD38 and transcription factor T-beT and TOX expression, suggesting a lower differentiation and depletion phenotype. Importantly, AKT inhibitionFormulation treatment resulted in ifnγ ⁺ TNFα ⁺ CD8 ⁺ T-cell frequency increases, which translates into increased cytotoxicity. AKT inhibitor treatment during ex vivo TIL amplification amplifies the proportion of less differentiated, more memory-like functional TILs.

Thus, the temporary inhibition of AKT signaling during TIL amplification represents a method for improving the efficacy of TIL products and amplifying the persistence and therapeutic efficacy of TIL in a clinical setting, including in combination with CCR and/or chemokine receptors. AKT inhibitor treated TIL maintained a higher frequency of CD69 with reduced TOX levels and increased cytokine output following stimulation ^- CD39 ^- And (3) cells. TIL treated with AKT inhibitors was observed to increase cytotoxic capacity in an allogeneic environment, which persisted even after repeated TIL stimulation. The AKT inhibitor treated TIL may be further modified to express more than one CCR or chemokine receptor as described elsewhere herein.

Example 22: use of non-genetic modifications to improve the phenotype of TIL products

In this example, the use of decitabine (a DNA hypomethylation agent) in culture as a non-genetic modifier of TIL products was probed. Decitabine may be combined with AKT inhibitors and CCR and chemokine receptors disclosed herein, as well as with other genetically modified TILs described herein. Patient tumors (n=8) of different tumor types (non-small cell lung cancer, head and neck cancer, ovarian cancer and breast cancer) were obtained from donors, minced and subjected to a 22 day amplification protocol for TIL production. Different doses (10 nM, 30nM and 100 nM) of decitabine were added to the culture before and during the REP phase or during REP alone during ex vivo amplification. The amplification potential and phenotypic and functional characteristics of TIL were assessed at the final TIL product.

The results shown in figure 76 demonstrate that decitabine treatment maintains TIL viability, but reduces expansion while increasing the cd4+/cd8+ T cell ratio. Figure 76 shows the expansion, viability and T cell distribution of control TIL and decitabine treated TIL. TIL is untreated (CTRL, grey bars) or treated with an increased concentration of decitabine. The treatment is during the REP phase only (blue bar) or both before REP and during REP (green Bars) are added. In FIG. 76, panel A shows the fold and viability of TIL at the end of the 22 day amplification process, while Panel B shows CD8 after the cryopreserved cell expansion process ⁺ 、CD4 ⁺ And frequency of cd4+ (foxp3+) cells (×p<0.05,**P<0.01). In FIG. 77, the results show that decitabine treatment during REP phase demonstrated an increase in CD8 ⁺ And CD4 ⁺ T in T cells _CM Frequency of sample cells. T cell subsets shown in control TIL and decitabine treated TIL, wherein CD8 ⁺ T of TIL after amplification _CM (CD45RA ^- CCR7 ⁺ )、T _EM (CD45RA ^- CCR7 ^- ) And T _EMRA (CD45 ⁺ CCR7 ^- ) The frequency of cells is shown in panel A, CD4 ⁺ TIL is shown in B plot (P)<0.05,**P<0.01). FIG. 78 shows the expression of surface markers on decitabine-treated TILs. Decitabine treatment was observed to increase CD8 ⁺ The frequency of co-stimulatory receptors on TIL, however, decreases inhibitory receptor expression. Panel A of FIG. 78 shows CD8 ⁺ Expression of CD25, ICOS, CD28 and IL-7R on TIL, however, panel B shows CD8 ⁺ Expression of inhibitory receptors PD-1 and TIGIT on TIL (P<0.05,**P<0.01,***P<0.001,****P<0.0001)。CD4 ⁺ Similar results were observed with TIL.

The expression of the decitabine treated TIL transcription factor is shown in fig. 79, which shows that decitabine treatment increases the expression of the memory-related transcription factor. The control or decitabine treated cryopreserved TIL was thawed and stained for flow cytometry analysis. Eomes, KLF2, BATF and T-bet at CD8 ⁺ Expression on TIL is shown in figure 79 (P)<0.05,**P<0.01). Cytokine expression following in vitro stimulation of control or decitabine treated TILs is presented in figure 80. Decitabine treatment was found to increase TNF-alpha and granzyme B-expressing CD8 following stimulation ⁺ Frequency of TIL. Cryopreserved control and decitabine treated TIL at 1:5 against cell scale anti-CD 3/CD28 beads were stimulated overnight. IFN-gamma, TNF-alpha and granzyme B in CD8 ⁺ Expression levels on TIL are shown in figure 80 (P)<0.05,**P<0.01)。

Controls and diesels were also evaluatedCytotoxicity of the TIL treated with the capecitabine. Fig. 81 shows that decitabine-treated TIL showed a sustained increase in cytotoxicity after repeated stimulation. In panel A, the control was cryopreserved and the TIL treated with 100nM decitabine at REP was compared toTHP-1 cells (Eurofins DiscoverX, friemont, california) at 10:1E: t cell ratios were co-cultured for 24 hours to measure cytotoxicity in an allogeneic environment. In panel B, control TIL and decitabine-treated TIL were stimulated every 5 days with transdcttm (Miltenyi Biotec, germany). One day after the third stimulation, cells were washed at 10:1 effector to target cell ratio KILR THP-1 cells were co-cultured for 24 hours to measure cytotoxicity. * P (P) <0.05。

FIG. 82 shows that decitabine-treated TIL shows reduced inhibitory receptor expression and lower levels of TOX with increased IL-7R expression following repeated stimulation. The phenotype of control and decitabine treated TILs was shown after repeated stimulation. Control TIL and decitabine-treated TIL were stimulated every 5 days with transdcttm (Miltenyi Biotec, germany). One day after the third stimulation, cells were washed and stained for flow cytometry analysis. In fig. 82, IL-7R, PD-1 and TIM3 expression of TIL after repeated stimulation is shown in panel a, whereas expression levels of transcription factors of TIL after repeated stimulation are shown in panel B (< 0.05, < 0.01).

In summary, decitabine treatment during TIL amplification can shift the equilibrium away from effector differentiation and toward more memory-like phenotypes. Treatment with 100nM decitabine during REP-only phase increases co-stimulatory receptor expression while decreasing inhibitory receptor expression. Decitabine treatment increases TNFa ⁺ IFN (interferon) gamma ray ⁺ TNFα ⁺ CD8 ⁺ The frequency of TIL simultaneously imparts increased killing activity, which persists even after repeated stimulation. Decitabine-treated TIL showed reduced TOX levels and lower frequency of PD1 after repeated stimulation ⁺ TIM3 ⁺ CD8 ⁺ TIL. This provides for inhibiting DNA methylation during TIL amplification to modify the non-geneticity of TIL Appearance to improve evidence of its therapeutic potential.

Example 23: double epitope TROP-2 and PD-L1 chimeric co-stimulatory receptors

In this example, additional bi-epitope CCR targeting TROP-2 and PD-L1 was prepared and tested using the procedure described above, as were PD-L1 scFv based on 38A1 and 19H9 and TROP-2 scFv based on cAR47A6 4 and KM 4097. These CCRs are designated CCR7.2, CCR8.2, CCR11.2 and CCR12.2. Suitable, non-limiting embodiments of CCRs which are prepared according to this example and which can be used as CCR constructs of the invention are shown in Table 87.

Table 87: the amino acid sequences of exemplary bi-epitope CCR designated CCR7.2, CCR8.2, CCR11.2 and CCR12.2

Suitable, non-limiting embodiments of nucleotides encoding CCRs prepared according to this example and useful as CCR constructs of the invention are shown in Table 88.

Table 88: nucleotide sequences of exemplary double epitope CCR designated CCR7.2, CCR8.2, CCR11.2 and CCR12

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Vectors encoding CCR designated CCR7.2, CCR8.2, CCR11.2 and CCR12.2 were prepared and presented in table 89.

Table 89: nucleotide sequences encoding exemplary vectors for the double epitope CCR designated CCR7.2, CCR8.2, CCR11.2 and CCR12.2

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Vector diagrams of exemplary vectors encoding the double epitope CCR designated CCR7.2, CCR8.2, CCR11.2 and CCR12.2 (corresponding to SEQ ID NO:685 to SEQ ID NO: 688) are presented in FIGS. 83 to 86.

The biological function of the CCR construct was tested with the Hek-IL-18SEAP reporter. HEKIL-18 reporter cells transduced with CCR8 and CCR8.2 (5X 10) ⁴ ) In the presence of anti-His antibodies (antibodies: protein ratio = 2:1, a step of; for example, for 5. Mu.g/mL PD-L1, 10. Mu.g/mL of anti-His antibody was added to stimulate with the indicated concentration of PD-L1 (His tag). After 24 hours, the supernatant was collected and SEAP levels were determined spectrophotometrically at 650nm after addition of the Quanti blue solution. The results are shown in FIG. 87. HEKIL-18 reporter cells transduced with CCR12 and CCR12.2 (5X 10) ⁴ ) In the presence of anti-His antibodies (antibodies: protein ratio = 2: 1) Stimulation was about TROP-2 (His tag) at the indicated concentrations. After 24 hours, the supernatant was collected and SEAP levels were determined spectrophotometrically at 650nm after addition of the Quanti blue solution. The results are shown in FIG. 88. The results of both sets of experiments were further compared to the IL-18 standard in FIG. 89 (in terms of PD-L1) and FIG. 90 (in terms of TROP-2), where an OD value of 0.5 was shown to correspond to approximately 3.175pg/mL IL-18 stimulation, showing custom activation.

Finally, TROP-2 expression on the cancer cell lines CaO-V3 and MCF-7 was assessed and quantified by flow through a PE fluorescence quantitative kit and compared to isotype controls. TROP-2 is expressed on CaO-V3 at a level of about 800,000 TROP-2 molecules per cell and on MCF-7 at a level of about 100,000 TROP-2 molecules per cell.

The foregoing examples are also embodiments of the present invention. Further embodiments of the invention include SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 and SEQ ID NO:680 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 or SEQ ID NO:680, has at least 99% identity to the sequence set forth in SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 or SEQ ID NO:680, has at least 98% identity to the sequence set forth in SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 or SEQ ID NO:680, has at least 97% identity to the sequence set forth in SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 and SEQ ID NO:680, has at least 96% identity to the sequence set forth in SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 and SEQ ID NO:680, has at least 95% identity to the sequence set forth in SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 and SEQ ID NO:680, has at least 90% identity to the sequence set forth in SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 and SEQ ID NO:680, or a sequence having at least 85% identity to SEQ ID NO: 677. SEQ ID NO: 678. SEQ ID NO:679 and SEQ ID NO:680 has an amino acid sequence having at least 80% identity.

Further embodiments of the invention include SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 has at least 99% identity to the sequence set forth in SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 has at least 98% identity to the sequence set forth in SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 has at least 97% identity to the sequence given in SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 has at least 96% identity to the sequence set forth in SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 has at least 95% identity to the sequence set forth in SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 has at least 90% identity to the sequence set forth in SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684, or a sequence having at least 85% identity to SEQ ID NO: 681. SEQ ID NO: 682. SEQ ID NO:683 and SEQ ID NO:684 has a nucleotide sequence with at least 80% identity.

Further embodiments of the invention include SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 has at least 99% identity to the sequence set forth in SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 has at least 98% identity to the sequence set forth in SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 has at least 97% identity to the sequence set forth in SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 has at least 96% identity to the sequence set forth in SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 has at least 95% identity to the sequence set forth in SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 has at least 90% identity to the sequence set forth in SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688, or a sequence having at least 85% identity to SEQ ID NO: 685. SEQ ID NO: 686. SEQ ID NO:687 and SEQ ID NO:688 has a nucleotide sequence with at least 80% identity.

Example 24: chimeric costimulatory receptors with 4-1BB intracellular domain

Additional CCR can be prepared using the methods described above. FIG. 91 shows CCR constructs designated CCR13, CCR14, CCR15 and CCR 16. Lentiviruses were prepared as described previously. Briefly, CCR was cloned into the pLenti-IRES-EGFP plasmid. AfeI/EcoRI enzyme recognition sequences were added on either side of the synthetic DNA sequence of CCR. Next, the complete DNA sequence was inserted into the pLenti-IRES-EGFP viral vector (CCR 13-CCR16 and CCR17-CCR19 in example 25). The pLenti virus vector is a self-inactivating lentiviral vector with gene expression driven by EF-1 alpha core promoter. To prepare lentiviruses, the pLenti vector and helper vector (VSV-G, gag/Pol) were co-transfected into 293T cells. Viral supernatants were collected from day 2 or 3 culture supernatants and then ultracentrifuged to concentrate lentiviruses for TIL transduction. The amino acid sequences of these CCRs are shown in Table 90.

Table 90: exemplary CCR amino acid sequences designated CCR13, CCR14, CCR15 and CCR16

Suitable, non-limiting embodiments of the nucleotides encoding CCR prepared according to this example and useful as CCR constructs of the invention are shown in table 91.

Table 91: exemplary CCR nucleotide sequences designated CCR13, CCR14, CCR15 and CCR16

Vectors encoding CCRs designated CCR13, CCR14, CCR15 and CCR16 were prepared as described above. The complete nucleotide sequences of these vectors are presented in table 92.

Table 92: nucleotide sequences encoding exemplary vectors for CCRs designated CCR13, CCR14, CCR15 and CCR16

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Vector diagrams of exemplary vectors encoding CCRs designated CCR13, CCR14, CCR15 and CCR16 (corresponding to SEQ ID NO:699 to SEQ ID NO: 702) are presented in FIGS. 92 to 95. These vectors were used to prepare lentiviral batches in this example.

After lentivirus preparation, TIL was transduced with each lentivirus batch, followed by 2 days of rest followed by 11 days REP amplification process. The surface expression of each CCR construct was detected by flow cytometry. The results are shown in FIG. 96.

Amplification, viability and killing efficacy of CCR-expressing REP TILs were also assessed. Prep TIL (n=3) was activated with Trans-ACT for 2 days, followed by transduction with lentiviral particle genes containing CCR constructs including Fas-4-1BB, PD-1-4-1BB, TGF-bRII-4-1BB, PD-1-28 (i.e., CCR13, CCR14, CCR15, and CCR 16) and control vector vectors. Two days after gene transduction, 3×10 was treated with 11 days REP amplification ⁴ And a prep TIL. FIG. 97 (A) shows fold expansion of TIL after CCR-expressing REP, (B) shows viability, and (C) shows passageKilling ability of TIL after CCR-expressing REP assessed by cytotoxicity assay. Briefly, let us (L)>THP-1 target cells (1.25X10) ⁴ ) And (3) withCCR transduced TIL cells (1.25×10 ⁵ E: t ratio = 10: 1) 100. Mu.L of CM2 medium containing 300IU/mL IL-2 was co-cultured in 96-well white plates. After 24 hours, 100 μl of KILR reagent was added to each well followed by 30 minutes incubation. Luminescence signals were measured to quantify dead cells. Percent killing was normalized based on control wells with cell lysis buffer added.

The foregoing examples are also embodiments of the present invention. Further embodiments of the invention include SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 has at least 99% identity to the sequence set forth in SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 has at least 98% identity to the sequence set forth in SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 has at least 97% identity to the sequence set forth in SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 has at least 96% identity to the sequence set forth in SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 has at least 95% identity to the sequence set forth in SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 has at least 90% identity to the sequence set forth in SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694, or a sequence having at least 85% identity to SEQ ID NO: 689. SEQ ID NO: 690. SEQ ID NO: 691. SEQ ID NO: 692. SEQ ID NO:693 and SEQ ID NO:694 has an amino acid sequence with at least 80% identity.

Further embodiments of the invention include SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 has at least 99% identity to the sequence set forth in SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 has at least 98% identity to the sequence set forth in SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 has at least 97% identity to the sequence set forth in SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 has at least 96% identity to the sequence set forth in SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 has at least 95% identity to the sequence set forth in SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 has at least 90% identity to the sequence set forth in SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698, or a sequence having at least 85% identity to SEQ ID NO: 695. SEQ ID NO: 696. SEQ ID NO:697 and SEQ ID NO:698 has a nucleotide sequence with at least 80% identity.

Further embodiments of the invention include SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 has at least 99% identity to the sequence given in SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 has at least 98% identity to the sequence given in SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 has at least 97% identity to the sequence given in SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 has at least 96% identity to the sequence given in SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 has at least 95% identity to the sequence given in SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 has at least 90% identity to the sequence given in SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702, or a sequence having at least 85% identity to SEQ ID NO: 699. SEQ ID NO: 700. SEQ ID NO:701 and SEQ ID NO:702 has a nucleotide sequence having at least 80% identity.

Example 25: chimeric costimulatory receptors with LTBR intracellular domains

Additional CCR can be prepared using the methods described above. FIG. 98 shows CCR constructs designated CCR17, CCR18 and CCR19 using LTBR intracellular domains. The amino acid sequences of these CCRs are shown in Table 93.

Table 93: exemplary CCR amino acid sequences designated CCR17, CCR18 and CCR19

Suitable, non-limiting embodiments of the nucleotides encoding CCR prepared according to this example and useful as CCR constructs of the present invention are shown in table 94.

Table 94: nucleotide sequences of exemplary CCRs designated CCR17, CCR18 and CCR19

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Vectors encoding CCRs designated CCR17, CCR18 and CCR19 were prepared as described above. The complete nucleotide sequences of these vectors are presented in table 95.

Table 95: nucleotide sequences of exemplary vectors encoding CCRs of CCR17, CCR18 and CCR19

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Vector diagrams of exemplary vectors encoding CCRs designated CCR17, CCR18 and CCR19 (corresponding to SEQ ID NO:709 to SEQ ID NO: 711) are presented in FIGS. 99 to 101. These vectors were used to prepare lentiviral batches in this example.

The foregoing examples are also embodiments of the present invention. Further embodiments of the invention include SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 or a conservative amino acid substitution thereof, or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 has at least 99% identity to the sequence set forth in SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 has at least 98% identity to the sequence set forth in SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 has at least 97% identity to the sequence given in SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 has at least 96% identity to the sequence set forth in SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 has at least 95% identity to the sequence set forth in SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 has at least 90% identity to the sequence set forth in SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705, or a sequence having at least 85% identity to SEQ ID NO: 703. SEQ ID NO:704 and SEQ ID NO:705 has an amino acid sequence with at least 80% identity.

Further embodiments of the invention include SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 has at least 99% identity to the sequence set forth in SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 has at least 98% identity to the sequence set forth in SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 has at least 97% identity to the sequence given in SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 has at least 96% identity to the sequence given in SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 has at least 95% identity to the sequence given in SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 has at least 90% identity to the sequence given in SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708, or a sequence having at least 85% identity to SEQ ID NO: 706. SEQ ID NO:707 and SEQ ID NO:708 has a nucleotide sequence having at least 80% identity.

Further embodiments of the invention include SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 has at least 99% identity to the sequence given in SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 has at least 98% identity to the sequence given in SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 has at least 97% identity to the sequence given in SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 has at least 96% identity to the sequence given in SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 has at least 95% identity to the sequence given in SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 has at least 90% identity to the sequence given in SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711, or has at least 85% identity to SEQ ID NO: 709. SEQ ID NO:710 and SEQ ID NO:711 has a nucleotide sequence having at least 80% identity.

Example 26: chimeric costimulatory receptors with ANTI-PD-L1 extracellular domain

Additional CCR can be prepared using the methods described above. These CCRs are designated CCR20, CCR21, CCR22, CCR23, CCR24 and CCR25 and use anti-PD-L1 19H9 extracellular domains. The amino acid sequences of these CCRs are shown in Table 96.

Table 96: exemplary CCR amino acid sequences designated CCR20, CCR21, CCR22, CCR23, CCR24 and CCR25

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Suitable, non-limiting embodiments of the nucleotides encoding CCR prepared according to this example and useful as CCR constructs of the invention are shown in table 97.

Table 97: exemplary CCR nucleotide sequences designated CCR20, CCR21, CCR22, CCR23, CCR24 and CCR25

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Vectors and procedures similar to those described in example 25 and example 26 can be used to prepare TILs expressing CCR20 to CCR 25.

The foregoing examples are also embodiments of the present invention. Further embodiments of the invention include SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717 or a conservative amino acid substitution thereof, or a fragment, variant, or derivative thereof, or a sequence that hybridizes to SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, has at least 99% identity to the sequence set forth in SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, has at least 98% identity to the sequence set forth in SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, has at least 97% identity to the sequence set forth in SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, has at least 96% identity to the sequence set forth in SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, has at least 95% identity to the sequence set forth in SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, has at least 90% identity to the sequence set forth in SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, or a sequence having at least 85% identity to SEQ ID NO: 712. SEQ ID NO: 713. SEQ ID NO: 714. SEQ ID NO: 715. SEQ ID NO:716 and SEQ ID NO:717, the sequence having at least 80% identity.

Further embodiments of the invention include SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 or a fragment, variant or derivative thereof, or a sequence identical to SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 has at least 99% identity to the sequence given in SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 has at least 98% identity to the sequence given in SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 has at least 97% identity to the sequence given in SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 has at least 96% identity to the sequence given in SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 has at least 95% identity to the sequence given in SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 has at least 90% identity to the sequence given in SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723, or a sequence having at least 85% identity to SEQ ID NO: 718. SEQ ID NO: 719. SEQ ID NO: 720. SEQ ID NO: 721. SEQ ID NO:722 and SEQ ID NO:723 has a nucleotide sequence having at least 80% identity.

***

The above examples are provided to give a complete disclosure and description of how to make and use embodiments of the compositions, processes, assays, systems and methods of the present invention to those of ordinary skill in the art and are not intended to limit the scope of what the inventors regard as their invention. Variations of the above modes for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims. All patents, patent applications, and publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains.

All headings and chapter designations are for clarity and reference purposes only and should not be construed as limiting the invention in any way. For example, those skilled in the art will appreciate the usefulness of combining the 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 as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

It will be apparent to those skilled in the art that many modifications and variations can be made thereto without departing from the spirit and scope of the application. The particular implementations and embodiments 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 by administering to a patient in need of treatment a population of tumor-infiltrating lymphocytes (TIL), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs), wherein the TIL, MILs, or PBLs are genetically modified to express a chimeric co-stimulatory receptor (CCR), the CCR comprising:

i. an extracellular domain;

hinge domain;

transmembrane domain; and

at least one intracellular domain.

2. The method of claim 1, wherein the cancer is treated by administering a population of TILs, the method comprising:

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

(d) Genetically modifying the second TIL population to express CCR;

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

3. The method of any one of claims 1-2, wherein the extracellular domain comprises a scFv binding domain.

4. The method of claim 3, wherein the scFv binding domain binds to a protein selected from the group consisting of CD19, CD20, CD22, CD24, CD33, CD38, CD39, CD73, CD123, CD138, CD228, LRRC15, CEA, fra, EPCAM, PD-L1, PSMA, gp100, MUC1, MCSP, EGFR, GD2, TROP-2, GPC3, MICA, MICB, VISTA, ULBP, HER2, MCM5, FAP, 5T4, LFA-1, B7-H3, IL-13 ra 2, FAS, tgfβ, tgfβrii, and MUC 16.

5. A method according to any one of claims 1 to 2, wherein the extracellular domain is selected from a PD-1 domain, a FAS domain and a tgfbetarii domain.

6. The method of any one of claims 1 to 5, wherein the intracellular domain is selected from the group consisting of CD28, CD134 (OX 40), CD278 (ICOS), CD137 (4-1 BB), CD27, CD40L, STAT3, IL-2rβ, IL-2rγ, IL-18R1, IL-18RAP, IL-7rα, IL-12R1, IL-12R2, IL-15rα, IL-21R, LTBR, and combinations thereof.

7. The method of any one of claims 1 to 6, wherein the transmembrane domain is selected from the group consisting of the transmembrane regions of CD3 a, CD3 β, cdζ, CD3 epsilon, CD4, CD5, CD8 a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, igG1, igG4, igD, IL-2 ra and CD 40L.

8. The method of any one of claims 2 to 7, wherein step (d) further comprises modifying TIL using a lentiviral gene to express CCR.

9. The method of any one of claims 1 to 8, wherein the TIL, MILs, or PBL is further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), SOCS1, ANKRD11, BCOR, and combinations thereof.

10. The method of any one of claims 2 to 9, wherein the cancer is a solid tumor cancer treated by administration of TIL.

11. The method of claim 10, wherein the cancer is selected from the group consisting of sarcoma, pancreatic cancer, liver cancer, glioblastoma, gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, lung cancer, non-small cell lung cancer, mesothelioma, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, kidney cancer, and renal cell carcinoma, and the patient is a human.

12. The method of claim 11, wherein the cancer is non-small cell lung cancer and the patient has at least one of:

(1) Tumor fraction (TPS) <1% of pre-determined PD-L1;

(2) PD-L1 tumor fraction (TPS) 1% to 49%; or (b)

(3) The absence of more than one driving mutation was determined in advance.

13. The method of claim 12, wherein the patient has a TPS of PD-L1 <1%.

14. The method of any one of claims 10 to 13, wherein the patient has a cancer that is not amenable to treatment by: EGFR inhibitors, BRAF inhibitors, ALK inhibitors, C-Ros inhibitors, RET inhibitors, ERBB2 inhibitors, BRCA inhibitors, MAP2K1 inhibitors, PIK3CA inhibitors, CDKN2A inhibitors, PTEN inhibitors, UMD inhibitors, NRAS inhibitors, KRAS inhibitors, NF1 inhibitors, MET inhibitors, TP53 inhibitors, CREBBP inhibitors, KMT2C inhibitors, KMT2D mutations, ARID1A mutations, RB1 inhibitors, ATM inhibitors, SETD2 inhibitors, FLT3 inhibitors, PTPN11 inhibitors, FGFR1 inhibitors, EP300 inhibitors, MYC inhibitors, EZH2 inhibitors, JAK2 inhibitors, xw7 inhibitors, CCND3 inhibitors, and GNA11 inhibitors.

15. The method of any one of claims 10 to 14, wherein the patient is free of more than one driving mutation.

16. The method of claim 15, wherein the one or more driving mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20, KRAS mutations, BRAF V600 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 splice 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.

17. The method of any one of claims 10 to 16, wherein the cancer is refractory or resistant to treatment by a chemotherapeutic agent or chemotherapy.

18. The method of any one of claims 10 to 17, wherein the cancer exhibits Sub>A refractory or resistance to treatment with Sub>A VEGF-Sub>A inhibitor.

19. The method of claim 18, wherein the VEGF-Sub>A inhibitor is selected from bevacizumab, lanbizumab, ai Lusu mab, and fragments, variants, and biological analogs thereof.

20. The method of any one of claims 10 to 19, wherein the cancer is refractory or resistant to treatment with a PD-1 inhibitor or a PD-L1 inhibitor.

21. The method of claim 20, wherein the PD-1 inhibitor or PD-L1 inhibitor is selected from nivolumab, pembrolizumab, cimab, tirelimumab, midodv Li Shan, terlipp Li Shan, dorimab, devaluzumab, esvaluzumab, atuzumab, refelsholt Li Shan, and fragments, variants, and biological analogs thereof.

22. The method of any one of claims 10 to 21, wherein the cancer is refractory or resistant to treatment with a CTLA-4 inhibitor.

23. The method of claim 22, wherein the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, za Li Fu limumab, and fragments, variants, and biological analogs thereof.

24. The method of any one of claims 10 to 23, wherein the IL-2 is initially present in the first and second cell culture media at an initial concentration of between 1000IU/mL and 6000 IU/mL.

25. The method of any one of claims 10 to 24, wherein the OKT-3 antibody is initially present in the second cell culture medium at an initial concentration of about 30 ng/mL.

26. The method of any one of claims 10 to 25, wherein the first or second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, 4-1BB agonist, OX-40 agonist, AKT inhibitor, and combinations thereof.

27. The method of any one of claims 10 to 26, wherein the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

28. The method of any one of claims 10 to 27, wherein the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third TIL population to the patient.

29. The method of claim 28, wherein the non-myeloablative lymphocyte depletion regimen comprises administering a dose of 60mg/m ² Cyclophosphamide/day for two days and then administered at a dose of 25mg/m ² Five days of fludarabine per day.

30. The method of claim 28, wherein the non-myeloablative lymphocyte depletion regimen comprises administering a dose of 60mg/m ² Cyclophosphamide per day and dose 25mg/m ² Fludarabine/day for two days and subsequent administration at a dose of 25mg/m ² Three days of fludarabine per day.

31. The method of any one of claims 10 to 30, wherein the method further comprises the step of beginning the treatment of the patient with the IL-2 regimen the next day after the third TIL population is administered to the patient.

32. The method of any one of claims 10 to 31, wherein the method further comprises the step of beginning the treatment of the patient with the IL-2 regimen on the same day as the third TIL population is administered to the patient.

33. The method of any one of claims 31 to 32, wherein the IL-2 regimen is a high dose IL-2 regimen comprising administering 600,000 or 720,000IU/kg of the aldesleukin, or fragment, variant or biological analog thereof, to a tolerizing bolus intravenous infusion every eight hours at 15 minutes.

34. The method of any one of claims 31-32, wherein the IL-2 regimen comprises administration of Bei Jiade Lu Jin or a fragment, variant or biological analog thereof.

35. The method of any one of claims 31-32, wherein the IL-2 regimen comprises administration of THOR-707 or a fragment, variant or biological analog thereof.

36. The method of any one of claims 31-32, wherein the IL-2 regimen comprises administering inner tile Lu Jin alpha or a fragment, variant or biological analog thereof.

37. The method of any one of claims 31 to 32, wherein the IL-2 regimen comprises administering an antibody or fragment, variant or biological analog thereof, said antibody 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.

38. The method of any one of claims 10 to 37, wherein a therapeutically effective population of TILs is administered, the therapeutically effective population of TILs comprising about 2 x 10 ⁹ Up to about 15X 10 ¹⁰ And TIL.

39. The method of any one of claims 10 to 38, wherein the first amplification is performed over a period of time less than 11 days.

40. The method of any one of claims 10 to 39, wherein the second amplification is performed over a period of time less than 11 days.

41. A composition comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs) genetically modified to express a chimeric co-stimulatory receptor (CCR), wherein the CCR comprises:

i. An extracellular domain;

hinge domain;

transmembrane domain; and

at least one intracellular domain.

42. The composition of claim 41, wherein the extracellular domain comprises an scFv binding domain.

43. The composition of claim 42, wherein the scFv binding domain is selected from the group consisting of an anti-CD 19 domain, an anti-CD 20 domain, an anti-CD 22 domain, an anti-CD 24 domain, an anti-CD 33 domain, an anti-CD 38 domain, an anti-CD 39 domain, an anti-CD 73 domain, an anti-CD 123 domain, an anti-CD 138 domain, an anti-CD 228 domain, an anti-LRRC 15 domain, an anti-CEA domain, an anti-fra domain, an anti-EPCAM domain, an anti-PD-L1 domain, an anti-PSMA domain, an anti-gp 100 domain, an anti-MUC 1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD 2 domain, an anti-TROP-2 domain, an anti-GPC 3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-MCM 5 domain, an anti-FAP domain, an anti-5T 4 domain, an anti-a-1 domain, an anti-B7-H3 domain, and an anti-MUC 16 domain.

44. A composition according to claim 41, wherein the extracellular domain is a PD-1 domain, a FAS domain or a TGF-beta RII domain.

45. The composition of any one of claims 41-44, wherein the intracellular domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, a STAT3 domain, an IL-2rβ domain, an IL-2rγ domain, an IL-18R1 domain, an IL-18RAP domain, an IL-7rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15rα domain, an IL-21R domain, and combinations thereof.

46. The composition of any one of claims 41 to 45, wherein the transmembrane domain is selected from the group consisting of a CD3 a domain, a CD3 β domain, a cdζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 a domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2 ra domain, an IL-2rβ domain, and an IL-2rγ domain.

47. The composition of any one of claims 41 to 46, wherein the TIL, MILs, or PBL is further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3) and combinations thereof.

48. A composition comprising a chimeric co-stimulatory receptor (CCR), wherein the CCR comprises:

i. an extracellular protein domain;

hinge protein domain;

transmembrane protein domain; and

at least one intracellular protein domain.

49. The composition of claim 48, wherein the extracellular protein domain comprises an scFv binding domain.

50. The composition of claim 49, wherein the scFv binding domain is selected from the group consisting of an anti-CD 19 domain, an anti-CD 20 domain, an anti-CD 22 domain, an anti-CD 24 domain, an anti-CD 33 domain, an anti-CD 38 domain, an anti-CD 39 domain, an anti-CD 73 domain, an anti-CD 123 domain, an anti-CD 138 domain, an anti-CD 228 domain, an anti-LRRC 15 domain, an anti-CEA domain, an anti-FR alpha domain, an anti-EPCAM domain, an anti-PD-L1 domain, an anti-PSMA domain, an anti-gp 100 domain, an anti-MUC 1 domain, an anti-MCSP domain, an anti-EGFR domain, an anti-GD 2 domain, an anti-TROP-2 domain, an anti-GPC 3 domain, an anti-MICA domain, an anti-MICB domain, an anti-VISTA domain, an anti-ULBP domain, an anti-5T 4 domain, an anti-A-1 domain, an anti-B7-H3 domain, an anti-IL-13, an anti-LFRalpha domain, and an anti-HER alpha domain.

51. The composition of claim 48, wherein the extracellular protein domain is a PD-1 domain, a FAS domain or a TGF-beta RII domain.

52. The composition of any one of claims 48 to 51, wherein the intracellular protein domain is selected from the group consisting of a CD28 domain, a CD134 (OX 40) domain, a CD278 (ICOS) domain, a CD137 (4-1 BB) domain, a CD27 domain, an IL-2rβ domain, an IL-2rγ domain, an IL-18R1 domain, an IL-18RAP domain, an IL-7rα domain, an IL-12R1 domain, an IL-12R2 domain, an IL-15rα domain, an IL-21R domain, and combinations thereof.

53. The composition of any one of claims 48 to 52, wherein the transmembrane protein domain is selected from the group consisting of a CD3 a domain, a CD3 β domain, a cdζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 a domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2 ra domain, an IL-2rβ domain, and an IL-2rγ domain.

54. The composition of any one of claims 48 to 53, wherein the hinge protein domain is selected from the group consisting of a CD3 a domain, a CD3 β domain, a CD ζ domain, a CD3 epsilon domain, a CD4 domain, a CD5 domain, a CD8 a domain, a CD9 domain, a CD16 domain, a CD22 domain, a CD27 domain, a CD28 domain, a CD33 domain, a CD37 domain, a CD45 domain, a CD64 domain, a CD80 domain, a CD86 domain, a CD134 domain, a CD137 domain, a CD154 domain, an IgG1 domain, an IgG4 domain, an IgD domain, an IL-2 ra domain, an IL-2rβ domain, and an IL-2rγ domain.

55. The composition of any one of claims 48 to 54, wherein the composition further comprises tumor infiltrating lymphocytes.

56. The composition of any one of claims 48 to 54, wherein the composition further comprises bone marrow infiltrating lymphocytes.

57. The composition of any one of claims 48 to 54, wherein the composition further comprises peripheral blood lymphocytes.

58. A method of treating cancer by administering to a patient in need of treatment a population of tumor-infiltrating lymphocytes (TIL), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs), wherein the TIL, MILs, or PBLs are genetically modified to express a chemokine receptor.

59. The method of claim 58, wherein the cancer is treated by administering a population of TILs, the method comprising:

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

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

60. The method of claim 59, wherein said chemokine receptor is a protein selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof.

61. The method of any one of claims 58 to 60, wherein step (d) further comprises modifying the TIL to express a chemokine receptor using a lentiviral or retroviral gene.

62. The method of any one of claims 58 to 61, wherein the TIL, MILs, or PBL is further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3), SOCS1, ANKRD11, BCOR, and combinations thereof.

63. The method of any one of claims 58 to 62, wherein the cancer is a solid tumor cancer treated by administration of TIL.

64. The method of claim 63, wherein the cancer is selected from the group consisting of sarcoma, pancreatic cancer, liver cancer, glioblastoma, gastrointestinal cancer, melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, lung cancer, non-small cell lung cancer, mesothelioma, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, kidney cancer, and renal cell carcinoma, and the patient is a human.

65. The method of claim 64, wherein the cancer is non-small cell lung cancer and the patient has at least one of:

(1) Tumor fraction (TPS) <1% of pre-determined PD-L1;

(2) PD-L1 tumor fraction (TPS) 1% to 49%; or (b)

(3) The absence of more than one driving mutation was determined in advance.

66. The method of claim 65, wherein the patient has a TPS of PD-L1 <1%.

67. The method of any one of claims 63-66, wherein the patient has a cancer that is not amenable to treatment by: EGFR inhibitors, BRAF inhibitors, ALK inhibitors, C-Ros inhibitors, RET inhibitors, ERBB2 inhibitors, BRCA inhibitors, MAP2K1 inhibitors, PIK3CA inhibitors, CDKN2A inhibitors, PTEN inhibitors, UMD inhibitors, NRAS inhibitors, KRAS inhibitors, NF1 inhibitors, MET inhibitors, TP53 inhibitors, CREBBP inhibitors, KMT2C inhibitors, KMT2D mutations, ARID1A mutations, RB1 inhibitors, ATM inhibitors, SETD2 inhibitors, FLT3 inhibitors, PTPN11 inhibitors, FGFR1 inhibitors, EP300 inhibitors, MYC inhibitors, EZH2 inhibitors, JAK2 inhibitors, xw7 inhibitors, CCND3 inhibitors, and GNA11 inhibitors.

68. The method of any one of claims 63-66, wherein the patient is not present with more than one driving mutation.

69. The method of claim 68, wherein the one or more driving mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20, KRAS mutations, BRAF V600 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 splice 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.

70. The method of any one of claims 63-69, wherein the cancer is refractory or resistant to treatment by a chemotherapeutic agent or chemotherapy.

71. The method of any one of claims 63-70, wherein the cancer exhibits refractory or resistant to treatment with Sub>A VEGF-Sub>A inhibitor.

72. The method of claim 71, wherein the VEGF-Sub>A inhibitor is selected from bevacizumab, lanbizumab, ai Lusu mab, and fragments, variants and biological analogs thereof.

73. The method of any one of claims 63-72, wherein the cancer exhibits refractory or resistant to treatment with a PD-1 inhibitor or a PD-L1 inhibitor.

74. The method of claim 73, wherein the PD-1 inhibitor or PD-L1 inhibitor is selected from nivolumab, pembrolizumab, cimab, tirelimumab, midodv Li Shan, terlipp Li Shan, dorimab, devaluzumab, esvaluzumab, atuzumab, refelsholt Li Shan, and fragments, variants, and biological analogs thereof.

75. The method of any one of claims 63 to 74, wherein the cancer is refractory or resistant to treatment with a CTLA-4 inhibitor.

76. The method of claim 75, wherein the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, za Li Fu limumab, and fragments, variants and biological analogs thereof.

77. The method of any one of claims 63-76, wherein the IL-2 is initially present in the first cell culture medium and the second cell culture medium at an initial concentration of between 1000IU/mL and 6000 IU/mL.

78. The method of any one of claims 63-77, wherein said OKT-3 antibody is initially present in the second cell culture medium at an initial concentration of about 30 ng/mL.

79. The method of any one of claims 63-78, wherein the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

80. The method of any one of claims 63-79, wherein the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

81. The method of any one of claims 63 to 80, wherein the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third TIL population to the patient.

82. The method of claim 81, wherein the non-myeloablative lymphocyte depletion regimen comprises administering a dose of 60mg/m ² Cyclophosphamide/day for two days and then administered at a dose of 25mg/m ² Five days of fludarabine per day.

83. The method of claim 82, wherein the non-myeloablative lymphocyte depletion regimen comprises administering a dose of 60mg/m ² Cyclophosphamide per day and dose 25mg/m ² Fludarabine/day for two days and subsequent administration at a dose of 25mg/m ² Three days of fludarabine per day.

84. The method of any one of claims 63-83, wherein the method further comprises the step of beginning the treatment of the patient with the IL-2 regimen the next day after the third TIL population is administered to the patient.

85. The method of any one of claims 63-83, wherein the method further comprises the step of beginning the treatment of the patient with the IL-2 regimen on the same day as the administration of the third TIL population to the patient.

86. The method of any one of claims 84-85, wherein the IL-2 regimen is a high dose IL-2 regimen comprising administering 600,000 or 720,000IU/kg of the aldesleukin, or fragment, variant or biological analog thereof, per eight hours at 15 minutes of bolus intravenous infusion until tolerizing.

87. The method of any one of claims 84-85, wherein the IL-2 regimen comprises administration of Bei Jiade Lu Jin or a fragment, variant or biological analogue thereof.

88. The method of any one of claims 84-85 wherein the IL-2 regimen comprises administration of THOR-707 or a fragment, variant or biological analog thereof.

89. The method of any one of claims 84-85 wherein the IL-2 regimen comprises administering inner tile Lu Jin alpha or a fragment, variant or biological analogue thereof.

90. The method of any one of claims 84-85, wherein the IL-2 regimen comprises administering an antibody or fragment, variant or biological analog thereof, said antibody 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.

91. The method of any one of claims 63-90, wherein a therapeutically effective population of TILs is administered, the therapeutically effective population of TILs comprising about 2 x 10 ⁹ Up to about 15X 10 ¹⁰ And TIL.

92. The method of any one of claims 63-91, wherein the first amplification is performed over a period of time less than 11 days.

93. The method of any one of claims 63-92, wherein the second amplification is performed over a period of time less than 11 days.

94. A composition comprising tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), or Peripheral Blood Lymphocytes (PBLs) genetically modified to express a chemokine receptor.

95. The composition of claim 94, wherein said chemokine receptor is a protein selected from the group consisting of CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7 (actr 3), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCR1, CX3CR1, and combinations thereof.

96. The composition of any one of claims 94 to 95, wherein the TIL, MILs, or PBL is further genetically modified to stabilize or temporarily reduce expression of a gene selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF βR2, PKA, CBL-B, BAFF (BR 3) and combinations thereof.

97. A composition comprising a chemokine receptor, wherein the composition further comprises tumor-infiltrating lymphocytes, bone marrow-infiltrating lymphocytes, or peripheral blood lymphocytes.