Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with a LAG-3 inhibitor and a PD-1 inhibitor

Abstract

A method for treating cancer in a patient in need thereof with TILs in combination with a LAG-3 inhibitor and a PD-1 / PD-L1 inhibitor is provided.

Description

Atorney Docket No. : 5133- WOTREATMENT OF CANCER PATIENTS WITH TUMOR INFILTRATING LYMPHOCYTE THERAPIES IN COMBINATION WITH A LAG-3 INHIBITOR AND A PD-1 INHIBITORBACKGROUND

[0001] Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are dominated by T cells, and IL-2-based TIL expansion followed by a “rapid expansion process” (REP) has become a preferred method for TIL expansion because of its 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. A number of approaches to improve responses to TIL therapy in melanoma and to expand TIL therapy to other tumor types have been explored with limited success, and the field remains challenging. Goff, et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al.. Clin. Cancer Res. 2011, / 7. 4550-57.

[0002] Immune checkpoint inhibitors are the first drugs to demonstrate improved survival rates in patients with advanced melanoma. Common immune checkpoint inhibitors include previously marketed CTLA-4 inhibitors and PD-1 inhibitors, as well as newly marketed LAG-3 inhibitor drugs. Ralli, et al., J. Immunol. Res. 2020. 9235638; Rashid, et al., Dermatol Clin. 2023, 41 (1), 49-63. Combination studies of TIL therapy with single immune checkpoint inhibitors have also been described, but further studies are ongoing and additional methods of treatment are needed (Kvemeland, et al., Oncotarget, 2020. 11(22), 2092-2105).

[0003] Furthermore, current TIL manufacturing and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, such that the potential to treat cancer patients has been severely limited. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes that are appropriate for use in treating cancer patients for whom very few or no viable treatment options remain. The present disclosure meets this need by providing manufacturing process for use in generating1DBl / 149983167.2Atorney Docket No. : 5133- WOTILs which can then be utilized for the treatment of cancer patients in combination with a PD-1 inhibitor and a LAG-3 inhibitor.BRIEF SUMMARY

[0004] Some embodiments disclosed herein provide a method of treating a cancer in a patient in need thereof, comprising: i) administering a therapeutic population of tumor infiltrating lymphocytes (TILs) to the patient; and ii) administering a LAG-3 inhibitor to the patient.

[0005] In some embodiments, the LAG-3 inhibitor is an anti -LAG-3 antibody. In some embodiments, the anti-LAG-3 antibody is one or more of relatlimab, favezelimab (MK- 4280), fianhmab, Sym022, GSK2831781 (IMP731), INCAGN02385, TSR-033, leramilimab (LAG525), or a biosimilar thereof. In some embodiments, the anti-LAG-3 antibody is relatlimab or a biosimilar thereof. In some embodiments, the anti-LAG-3 antibody is favezelimab (MK-4280) or a biosimilar thereof. In some embodiments, the anti-LAG-3 antibody is fianlimab or a biosimilar thereof. In some embodiments, the method further comprises administering a PD-1 inhibitor and / or a PD-L1 inhibitor to the patient. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll antibody. In some embodiments, the anti-PD-1 antibody or the anti-PD-Ll antibody is one or more of nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), cemiplimab (LIBTAY O®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), durvalumab (IMFINZI®), or a biosimilar thereof. In some embodiments, the anti-PD-1 antibody is nivolumab or a biosimilar thereof. In some embodiments, the anti- PD-1 antibody is pembrolizumab or a biosimilar thereof. In some embodiments, the anti-PD- 1 antibody is cemiplimab or a biosimilar thereof. In some embodiments, the anti-LAG-3 antibody is relatlimab or a biosimilar thereof and the anti-PD-1 antibody is nivolumab or a biosimilar thereof. In some embodiments, relatlimab or a biosimilar thereof is administered at a dosage of about 160 mg and nivolumab or a biosimilar thereof is administered at a dosage of about 480 mg. infused over 30 minutes. In some embodiments, relatlimab or a biosimilar thereof and nivolumab or a biosimilar thereof are administered weekly, once every two weeks, or once every' three weeks. In some embodiments, the LAG-3 inhibitor and / or the PD- 1 / PD-L1 inhibitor is administered contemporaneously with the population of TILs. In someAtorney Docket No. : 5133- WO embodiments, the LAG-3 inhibitor and / or the PD-1 / PD-L1 inhibitor is not administered contemporaneously with the population of TILs. In some embodiments, the LAG-3 inhibitor and / or the PD-1 / PD-L1 inhibitor is administered after administering the population of TILs. In some embodiments, the LAG-3 inhibitor and / or the PD-1 / PD-L1 inhibitor is administered at least one week after administering the population of TILs. In some embodiments, the LAG-3 inhibitor and / or the PD-1 / PD-L1 inhibitor is maintained after administering the population of TILs. In some embodiments, the patient is refractory to pre-treatment with a LAG-3 inhibitor or a biosimilar thereof. In some embodiments, the patient is refractor}' to pre-treatment with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof. In some embodiments, the patient has been previously treated with a PD-1 inhibitor or a biosimilar thereof. In some embodiments, the PD-1 inhibitor is selected from the group consisting of nivolumab. pembrolizumab. cemiplimab, and biosimilars thereof. In some embodiments, the patient has been previously treated with a PD-L1 inhibitor or a biosimilar thereof. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of avelumab, atezolizumab, durvalumab, and biosimilars thereof. In some embodiments, the patient is naive to an immune checkpoint inhibitor (ICI) treatment. In some embodiments, the ICI treatment comprises a PD-1 inhibitor and / or a PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor and / or PD-L1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, avelumab, atezolizumab, durvalumab, and biosimilars thereof. In some embodiments, the ICI treatment comprises a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is selected from the group consisting of relatlimab, favezelimab (MK- 4280), fianlimab, Sym022, GSK2831781 (IMP731), INCAGN02385, TSR-033, leramilimab (LAG525), and a biosimilar thereof. In some embodiments, the ICI treatment comprises a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is selected from the group consisting of ipilimumab, tremelimumab, zalifrelimab, AGEN1181, BMS-986218. BCD-145, ONC-392, CS1002, REGN4659, ADG1 16, and a biosimilar thereof. In some embodiments, the patient has been previously treated with a chemotherapeutic regimen. In some embodiments, the chemotherapeutic regimen comprises carboplatin, paclitaxel, pemetrexed, and / or cisplatin. In some embodiments, the patient has not been previously treated with a chemotherapeutic regimen. In some embodiments, the patient has been previously treated with an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitor is bevacizumab. In some embodiments, the patient has not been previously treated with an angiogenesis inhibitor.Atorney Docket No. : 5133- WO

[0006] Some embodiments disclosed herein provide a method of treating a cancer in a patient in need thereof comprising: i) administering a therapeutic population of tumor infiltrating lymphocytes (TILs) to the patient; and ii) administering a LAG-3 inhibitor to the patient, wherein the therapeutic population of TILs is produced by:(a) obtaining and / or receiving a first population of TILs from a tumor resected from the patient;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

[0007] In some embodiments, the method further comprises: (d) harvesting therapeutic population of TILs. In some embodiments, the method further comprises: (e) transferring the harvested therapeutic population of TILs into an infusion bag. In some embodiments, the method further comprises: (f) cry opreserving the infusion bag comprising the harvested therapeutic population of TILs using a cry opreservation process. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 11 days. In some embodiments, the first expansion is performed over a period of about 11 days, and the second expansion is performed over a period of about 11 days. In some embodiments, step (b) and step (c) are performed in a closed system, wherein the transition from step (b) to step (c) occurs without opening the closed system. In some embodiments, the transition from step (c) to step (d) occurs without opening the closed system. In some embodiments, the transition from step (d) to step (e) occurs without opening the closed system.Atorney Docket No. : 5133- WO

[0008] In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for five days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting on the day after the administration of the TILs to the patient. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting on the same day as administration of the TILs to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 lU / kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15 -minute bolus intravenous infusion every eight hours until tolerance. In some embodiments, a therapeutically effective population of TILs is administered and comprises from about 1 xlO9to about 10*1010TILs. In some embodiments, a therapeutically effective population of TILs is administered and comprises from about 7.5x l09to about 7.5xlO10TILs. In some embodiments, the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypopharynx cancer, larynx cancer, nasopharynx cancer, oropharynx cancer, and pharynx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penileAtorney Docket No. : 5133- WO cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ew ing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1: Exemplary Gen 2 (process 2A) chart providing an overview' of Steps A through F.

[0010] Figures 2A-2C: Process flow chart of an embodiment of Gen 2 (process 2A) for TIL manufacturing.

[0011] Figure 3: Process flowchart for Cohort ID.

[0012] Figures 4A-4D: Schedule of Assessments for Cohort ID (Melanoma) receiving lifileucel in combination with nivolumab-relatlimab.DETAILED DESCRIPTIONI. Definitions

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

[0014] The terms “co-administration,” “co-administering,” '‘administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the present disclosure, for example, a plurality of TILs) to a subject so that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0015] The term “in vivo” refers to an event that takes place in a subject's body.Atorney Docket No. : 5133- WO

[0016] The term “z vitro’' refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0017] The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject’s body. Aptly, the cell, tissue and / or organ may be returned to the subject’s body in a method of surgery' or treatment.

[0018] By “population of cells” (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from 1 X 106to 1 X 1010or more in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1 x 108cells. REP expansion is generally done to provide populations of 1.5 x 109to 1.5 x io10cells for infusion.

[0019] By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about -150 °C to -60 °C. General methods for cry opreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.

[0020] By “thawed cryopreserved TILs” herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.

[0021] The term “cryopreservation media” or “cryopreservation medium” refers to any medium that can be used for cry opreservation of cells. Such media can include media comprising 7% to 10% DMSO. Exemplary media include CRYOSTOR®CS10, Hyperthermasol, as well as combinations thereof. The term “CS10” refers to a cryopreservation medium which is obtained from Stemcell Technologies or from Biolife Solutions. The CS10 medium may be referred to by the trade name “CRYOSTOR® CS10”. The CS 10 medium is a serum-free, animal component-free medium which comprises DMSO. In some embodiments, the CS10 medium comprises 10% DMSO.Atorney Docket No. : 5133- WO

[0022] The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present disclosure. Closed systems include, for example, but are not limited to, closed G- containers. Once a tumor segment is added to the closed system, the system is not opened to the outside environment until the TILs are ready to be administered to the patient.

[0023] The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.

[0024] The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g.. a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti- CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3e. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0025] The term “OKT-3” (also referred to herein as “0KT3”) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (EC ACC) and assigned Catalogue No. 86022706.

[0026] The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by reference herein. For example, the term IL-2 encompasses human, recombinant forms of IL-2 such asAtorney Docket No. : 5133- WO aldesleukin (PROLEUKIN®, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix. Inc., Portsmouth. NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, N , USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl- 1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug bempegaldesleukin (NKTR-214, pegylated human recombinant IL-2 in which an average of 6 lysine residues are N6substituted with [(2,7- bis { [methylpoly (oxy ethylene)] carbamoyl} -9H-fluoren-9-yl)methoxy]carbonyl), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or which 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 Al or the method described in Example 1 of U.S. Patent Application Publication No. US 2019 / 0275133 Al, the disclosures of which are incorporated by reference herein. Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use as described herein are described in U.S. Patent Application Publication No. US 2014 / 0328791 Al and International Patent Application Publication No. WO 2012 / 065086 Al, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use as described herein are described in U.S. Patent Nos. 4,766,106. 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use as described herein are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated by reference herein.

[0027] In some embodiments, an IL-2 form suitable for use as described herein is THOR- 707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use as described herein are described in U.S. Patent Application Publication Nos. US 2020 / 0181220 Al and US 2020 / 0330601 Al, the disclosures of which are incorporated by reference herein. In some embodiments, and IL-2 form suitable for use as described herein is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35. T37, R38, T41. F42, K43, F44, Y45, E61 , E62, E68, K64, P65, V69, L72, and Y 107. In some embodiments, theAtorney Docket No. : 5133- WO conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use as described herein is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use as described herein is pegylated as disclosed in U.S. Patent Application Publication No. US 2020 / 0181220 Al and U.S. Patent Application Publication No. US 2020 / 0330601 AL

[0028] In some embodiments, an IL-2 form suitable for use as described herein is nemvaleukin alfa, also known as ALKS-4230, which is available from Alkermes, Inc. Nemvaleukin alfa is also known as human interleukin 2 fragment (1-59), variant (Cys125>Ser51), fused via peptidyl linker (60GG61) to human interleukin 2 fragment (62-132), fused via peptidyl linker (133GSGGGS138) to human interleukin 2 receptor a-chain fragment (139-303), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys125(51)>Ser] -mutant (1-59), fused via a G2 peptide linker (60- 61) to human interleukin 2 (IL-2) (4-74)-peptide (62-132) and via a GSGsS peptide linker (133-138) to human interleukin 2 receptor a-chain (IL2R subunit alpha, IL2Ra, IL2RA) (1- 165)-peptide (139-303), produced in Chinese hamster ovary’ (CHO) cells, glycoform alfa. The preparation and properties of nemvaleukin alfa. as well as additional alternative forms of IL-2, is described in U.S. Patent Application Publication No. US 2021 / 0038684 Al and U.S. Patent No. 10,183,979, the disclosures of which are incorporated by reference herein. Other IL-2 forms suitable for use as described herein are described in U.S. Patent No. 10.183,979, the disclosures of which are incorporated by reference herein. Optionally, in some embodiments, an IL-2 form is a fusion protein comprising a first fusion partner that is linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-IRa or a protein having at least 98% amino acid sequence identity to IL-IRa and having the receptor antagonist activity of IL-Ra, and wherein the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc regionand wherein the half-life of the fusion protein is improved as compared to a fusion of the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker.

[0029] In some embodiments, an IL-2 form suitable for use as described herein includes an antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chainAtorney Docket No. : 5133- WO variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3: and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL. wherein the IL-2 molecule is a mutein, and wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulator}' T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. US 2020 / 0270334 Al, the disclosures of which are incorporated by reference herein. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulator}' T cells.

[0030] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some instances, the IL-2 mutein comprising an R67A substitution. In some embodiments, the IL-2 mutein comprises an amino acid sequence in Table 1 in U.S. Patent Application Publication No. US 2020 / 0270334 AL the disclosure of which is incorporated by reference herein.

[0031] In some embodiments, the antibody components of the antibody cytokine engrafted protein described herein comprise immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine engrafted protein described herein has a longer serum half-life than a wild-type IL-2 molecule such as, but not limited to, aldesleukin or a comparable molecule.

[0032] The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (ThO cells) to Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2. 66-70. Upon activation by IL-4. Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgGi expression from BAtorney Docket No. : 5133- WO cells. Recombinant human IL-4 suitable for use as described herein is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ. USA (Cat. No. CYT-211) and ThermoFisher Scientific. Inc., Waltham. MA, USA (human IL- 15 recombinant protein. Cat. No. Gibco CTP0043).

[0033] The term ‘IL-7’’ (also referred to herein as “IL7”) refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fr ' and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use as described herein is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific. Inc., Waltham. MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071).

[0034] The term "IL- 15“ (also referred to herein as “IL15”) refers to the T cell growth factor know n as interleukin- 15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri. Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares (3 and y 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) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick. NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific. Inc.. Waltham. MA. USA (human IL-15 recombinant protein, Cat. No. 34-8159-82).

[0035] The term '‘IL-21’’ (also referred to herein as '‘IL21”) refers to the pleiotropic cytokine protein know n as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95. the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+T cells. Recombinant human IL- 21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with aAtorney Docket No. : 5133- WO molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA. USA (human IL-21 recombinant protein, Cat. No. 14-8219-80).

[0036] The term “LAG-3” refers to the lymphocyte activation gene-3 protein, an immune checkpoint receptor or T cell co-inhibitor, also known as CD223. The amino acid sequence of full-length LAG-3 is provided in GenBank as accession number NP 002277.4. LAG-3 is a member of the immunoglobulin (Ig) superfamily. LAG-3 is a 503-amino acid type-1 transmembrane protein with four extracellular Ig-like domains DI to D4 and is expressed on activated T cells, natural killer cells, B cells, plasmacytoid dendritic cells, and regulatory T cells. The LAG-3 receptor binds to MHC class II molecules present on antigen presenting cells (APCs).

[0037] When “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age. weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the tumor infiltrating lymphocytes (e.g. secondary TILs or genetically modified cytotoxic lymphocytes) described herein may be administered at a dosage of 104to I011cells / kg body weight (e.g., 105to 106, 105to 1010, 105to 1011, 106to IO10, 106to 10n,107to 1011, 107to 1010, 108to 1011, 108to 1010, 109to 1011, or 109to 1010cells / kg body weight), including all integer values within those ranges. TILs (including in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. The TILs (including, in some cases, genetically engineered TILs) can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg, et al.. New Eng. J. of Med. 1988, 319, 1676). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0038] The term “microenvironment,” as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cuesAtorney Docket No. : 5133- WO that promote neoplastic transformation, support tumor grow th and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,’" as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment.

[0039] In some embodiments, the present disclosure includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated w ith non-myeloablative chemotherapy prior to an infusion of TILs. In some embodiments, the population of TILs may be provided wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of TILs. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion (at day 0), the patient receives an intravenous infusion of IL-2 at 720,000 lU / kg every 8 hours. In some embodiments, the patient receives an intravenous infusion of IL-2 at 720,000 lU / kg every 8 hours to physiologic tolerance.

[0040] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system ("‘cytokine sinks”). Accordingly, some embodiments utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the TILs.

[0041] The term “effective amount” or “therapeutically effective amount” refers to that amount of an agent (e.g., cells, antibody, or other compound) or combination of agents as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration. The term also applies to a dose that will induce a particular response in target cells. The specific dose will vary depending on the particular agent(s) chosen, the dosing regimen to be followed, whether the agent is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery' system in which the agent is carried.Atorney Docket No. : 5133- WO

[0042] The terms ‘"treatment”, “treating”, “treat”, and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment”, as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery7of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g. , in the case of a vaccine.

[0043] The terms “non-myeloablative chemotherapy,” “non-myeloablative lymphodepletion,” “NMALD,” “NMA LD,” “NMA-LD,” and any variants of the foregoing, are used interchangeably to indicate a chemotherapeutic regimen designed to deplete the patient's lymphoid immune cells while avoiding depletion of the patient's myeloid immune cells. Typically, the patient receives a course of non-myeloablative chemotherapy prior to the administration of tumor infiltrating lymphocytes to the patient as described herein.

[0044] The term “heterologous” when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid may be recombinantly produced, having two or more sequences from unrelated genes arranged to make anew functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0045] The terms “sequence identity,” “percent identity,” and “sequence percent identity” (or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary ) for maximum correspondence, not consideringAtorney Docket No. : 5133- WO any conservative amino acid substitutions as part of the sequence identity7. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity7include for example the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either 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 (Genentech, South San Francisco, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0046] As used herein, the term ’‘variant” encompasses but is not limited to proteins, antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference protein by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference protein. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference protein. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. A variant retains one or more functions of the reference protein, e.g., the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0047] By "tumor infiltrating lymphocytes” or "TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Thl and Thl7 CD4+T cells, natural killer cells, dendritic cells and Ml macrophages. TILs include both primary and secondary TILs. "Primary7TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs ("REP TILs”) as well as “reREP TILs” as discussed herein. reREP TILs can include forAtorney Docket No. : 5133- WO example second expansion TILs or second additional expansion TILs. TIL cell populations can include genetically modified TILs.

[0048] TILs can generally be defined either biochemically , using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR a(3, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability' to infiltrate solid tumors upon reintroduction into a patient. TILs may further be characterized by potency - for example, TILs may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. TILs may be considered potent if, for example, interferon (IFNy) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, greater than about 1000 pg / mL.

[0049] The terms “pharmaceutically acceptable carrier" or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in therapeutic compositions disclosed herein is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0050] The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 10%, within 5%, or within 1% of a given value or range. The allow able variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may beAtorney Docket No. : 5133- WO 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. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is "about" or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0051] The transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any. are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of’ limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

[0052] The terms “antibody” and its plural form “antibodies” refer to whole immunoglobulins and any antigen-binding fragment (“antigen-binding portion”) or single chains thereof. An “antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and tw o light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy’ chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions of an antibody may be further subdivided into regions of hyperv an ability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1. FR2. CDR2, FR3, CDR3, FR4. TheAtorney Docket No. : 5133- WO variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g, effector cells) and the first component (Clq) of the classical complement system.

[0053] The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility7complex (MHC) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.

[0054] The terms “monoclonal antibody,” “mAb,” “monoclonal antibody composition,” or their plural forms refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific to certain receptors can be made using knowledge and skill in the art of injecting test subjects with suitable antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.

[0055] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “fragment”), as used herein, refers to one or more fragments ofAtorney Docket No. : 5133- WO an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VII, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment (Ward, et al., Nature. 1989, 341, 544-546), which may consist of a VH or a VL domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH. are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH 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 within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. In some embodiments, a scFv protein domain comprises a VH portion and a VL portion. A scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH 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) and R. E. Bird and B. W. Walker. Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991), the disclosures of which are incorporated by reference herein.

[0056] The term “human antibody,” as used herein, is intended to include antibodies having variable regions in which both the framew ork and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specificAtorney Docket No. : 5133- WO mutagenesis in vitro or by somatic mutation in vivo). 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, such as a mouse, have been grafted onto human framework sequences.

[0057] The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g, a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0058] The term “recombinant human antibody”, as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library', and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0059] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

[0060] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”Atorney Docket No. : 5133- WO

[0061] The term “human antibody derivatives” refers to any modified form of the human antibody, including a conjugate of the antibody and another active pharmaceutical ingredient or antibody. The terms “conjugate,” “antibody-drug conjugate”, “ADC,” or “immunoconjugate” refers to an antibody, or a fragment thereof, conjugated to another therapeutic moiety, which can be conjugated to antibodies described herein using methods available in the art.

[0062] The terms “humanized antibody,” “humanized antibodies,” and “humanized” are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two. variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), ty pically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525;Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein may also be modified to employ any Fc variant which is known to impart an improvement (e.g., reduction) in effector function and / or FcR binding. The Fc variants may include, for example, any one of the amino acid substitutions disclosed in International Patent Application Publication Nos. WO 1988 / 07089 Al, WO 1996 / 14339 Al , WO 1998 / 05787 Al , WO 1998 / 23289 A 1 , WO 1999 / 51642 Al , WOAtorney Docket No. : 5133- WO99 / 58572 Al, WO 2000 / 09560 A2, WO 2000 / 32767 Al, 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 / 063351 A2. WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 Al, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 Al, WO 2006 / 047350 A2, and WO 2006 / 085967 A2; and U.S. Patent Nos. 5,648,260; 5,739,277; 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 disclosures of which are incorporated by reference herein.

[0063] The term '‘chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as 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.

[0064] A “diabody” is a small antibody fragment with two antigen-binding sites. The fragments comprises a heavy chain variable domain (Vn) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g, European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161; and Bolliger, et al.. Proc. Natl. Acad. Set. USA 1993, 90, 6444-6448.

[0065] The term “glycosylation” refers to a modified derivative of an antibody. An aglycoslated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Aglycosylation may increase the affinity of the antibody for antigen, as described in U.S. Patent Nos. 5,714.350 and 6,350,861. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody havingAtorney Docket No. : 5133- WO increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosy Itransferase gene, FUT8 (alpha (1,6) fucosy Itransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by the targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see e.g. U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki. et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example. European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO 03 / 035835 describes a variant CHO cell line, Lee 13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, et al.. J. Biol. Chem. 2002, 277, 26733-26740. International Patent Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0066] "Pegylalion" refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody orAtorney Docket No. : 5133- WO antibody fragment. Peg lation may, for example, increase the biological (e.g., serum) half life of the antibody. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci-Cio)alkoxy- or aryloxy -poly ethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies, as described for example in European Patent Nos. EP 0154316 and EP 0401384 and U.S. Patent No. 5,824,778, the disclosures of each of which are incorporated by reference herein.

[0067] The term “biosimilar” means a biological product, including a monoclonal antibody or protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN®), a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof’ of aldesleukin. In Europe, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The already authorized original biological medicinal product may be referred to as a “reference medicinalAtorney Docket No. : 5133- WO product” in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity', mechanism of action, safety profiles and / or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality7characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety7profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and / or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity7of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to. glycosylation, oxidation, deamidation, and / or truncation which is / are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and / or efficacy of the medicinal product. The biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although notAtorney Docket No. : 5133- WO exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and / or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in, for example, pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies.II. Gen 2 TIL Manufacturing Processes

[0068] An exemplary' family of TIL processes known as Gen 2 (also known as process 2A) containing some of these features is depicted in Figures 1 and 2A-2C. An embodiment of Gen 2 is shown in Figures 2A-2C.

[0069] As discussed herein, the method can include a step relating to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplant into a patient, and methods of testing said metabolic health. As generally outlined herein, TILs are generally taken from a patient sample and manipulated to expand their number prior to transplant into a patient. In some embodiments, the TILs may be optionally genetically manipulated as discussed below.

[0070] In some embodiments, the TILs may be cryopreserved. Once thawed, they may also be restimulated to increase their metabolism prior to infusion into a patient.

[0071] In some embodiments, the first expansion (including processes referred to as the pre-REP as well as processes shown in Figure 1 as Step A) is shortened to 3 to 14 days and the second expansion (including processes referred to as the REP as well as processes shown in Figure 1 as Step B) is shorted to 7 to 14 days, as discussed in detail below as well as in the examples and figures. In some embodiments, the first expansion (for example, an expansion described as Step B in Figure 1) is shortened to 11 days and the second expansion (forAtorney Docket No. : 5133- WO example, an expansion as described in Step D in Figure 1) is shortened to 11 days. In some embodiments, the combination of the first expansion and second expansion (for example, expansions described as Step B and Step D in Figure 1) is shortened to 22 days, as discussed in detail below and in the examples and figures.

[0072] The “Step” Designations A, B, C, etc., below are in reference to Figure 1 and in reference to certain embodiments described herein. The ordering of the Steps below and in Figure 1 is exemplary and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps is contemplated by the present application and the methods disclosed herein.A. STEP A: Obtain Patient Tumor Sample

[0073] In general, TILs are 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 evaluated for phenotype and metabolic parameters as an indication of TIL health.

[0074] A patient tumor sample may be obtained using methods know n in the art. generally via surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells. In some embodiments, multilesional sampling is used. In some embodiments, surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells includes multilesional sampling (z.e., obtaining samples from one or more tumor sites and / or locations in the patient, as well as one or more tumors in the same location or in close proximity)- In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of lung tissue. In some embodiments, useful TILs are obtained from non-small cell lung carcinoma (NSCLC). The solid tumor may be of skin tissue. In some embodiments, useful TILs are obtained from a melanoma.

[0075] Once obtained, the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm3, with from about 2-3 mm3being particularly useful. In some embodiments, the TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests may be produced by incubation in enzymatic media (e.g.,Atorney Docket No. : 5133- WORoswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicine, 30 units / mL of DNase and 1.0 mg / mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 Al, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.

[0076] Tumor dissociating enzyme mixtures can include one or more dissociating (digesting) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), ACCUTASE™, ACCUMAX™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase. elastase, papain, protease type XIV (pronase), deoxyribonuclease 1 (DNase), trypsin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof.

[0077] In some embodiments, the dissociating enzymes are reconstituted from lyophilized enzymes. In some embodiments, lyophilized enzy mes are reconstituted in an amount of sterile buffer such as Hanks' Balanced Salt Solution (HBSS).

[0078] In some instances, collagenase (such as animal free- ty pe 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL buffer. In some embodiment, after reconstitution the collagenase stock ranges from about 100 PZ U / mL-about 400 PZ U / mL, e.g., about 100 PZ U / mL-about 400 PZ U / mL, about 100 PZ U / mL-about 350 PZ U / mL, about 100 PZ U / mL-about 300 PZ U / mL, about 150 PZ U / mL-about 400 PZ U / mL, about 100 PZ U / mL, about 150 PZ U / mL, about 200 PZ U / mL. about 210 PZ U / mL, about 220 PZ U / mL, about 230 PZ U / mL, about 240 PZ U / mL, about 250 PZ U / mL, about 260 PZ U / mL, about 270 PZ U / mL, aboutAtorney Docket No. : 5133- WO280 PZ U / mL, about 289.2 PZ U / mL, about 300 PZ U / mL, about 350 PZ U / mL, or about 400 PZ U / mL.

[0079] In some embodiments, neutral protease is reconstituted in 1 m of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC U / vial. In some embodiments, after reconstitution the neutral protease stock ranges from about 100 DMC / mL-about 400 DMC / mL, e.g., about 100 DMC / mL-about 400 DMC / rnL, about 100 DMC / mL-about 350 DMC / mL, about 100 DMC / mL-about 300 DMC / mL, about 150 DMC / mL-about 400 DMC / mL, about 100 DMC / mL, about 110 DMC / mL, about 120 DMC / mL, about 130 DMC / mL. about 140 DMC / mL. about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL.

[0080] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, after reconstitution the DNase I stock ranges from about 1 KU / mL-10 KU / mL, e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL.

[0081] In some embodiments, the stock of enzymes is variable and the concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock can be verified. In some embodiments, the final amount of enzyme added to the digest cocktail is adjusted based on the determined stock concentration.

[0082] In some embodiments, the enzy me mixture includes about 10.2-ul of neutral protease (0.36 DMC U / mL), 21.3 pL of collagenase (1.2 PZ / mL) and 250-ul of DNAse I (200 U / mL) in about 4.7 mL of sterile HBSS.

[0083] As indicated above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumors are not fragmented. In some embodiments, the solid tumors are not fragmented and are subjected to enzy matic digestion as whole tumors. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase,Atorney Docket No. : 5133- WODNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO2. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase. DNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO2 with rotation. In some embodiments, the tumors are digested overnight with constant rotation. In some embodiments, the tumors are digested overnight at 37°C, 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzy mes to form a tumor digest reaction mixture.

[0084] In some embodiments, the tumor is reconstituted with the lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0085] In some embodiments, the enzy me mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock for the collagenase is a 100 mg / mL 10X working stock.

[0086] In some embodiments, the enzy me mixture comprises DNAse. In some embodiments, the working stock for the DNAse is a 10,000 TU / mL 1 OX working stock.

[0087] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock for the hyaluronidase is a 10 mg / mL 10X working stock.

[0088] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 lU / mL DNAse, and 1 mg / mL hyaluronidase.

[0089] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 lU / mL DNAse, and 1 mg / mL hyaluronidase.

[0090] In general, the harvested cell suspension is called a “primary' cell population” or a “freshly harvested” cell population.

[0091] In some embodiments, fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is dissection. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or fragmenting a tumor sample obtained from a patient.

[0092] In some embodiments, where the tumor is a solid tumor, the tumor undergoes physical fragmentation after the tumor sample is obtained in, for example, Step A (as provided in Figure 1). In some embodiments, the fragmentation occurs beforeAtorney Docket No. : 5133- WO cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after obtaining the tumor and in the absence of any cry opreservation. In some embodiments, the tumor is fragmented and 10, 20, 30. 40 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm3. In some embodiments, the multiple fragments comprise about 30 to about 60 fragments with a total volume of about 1300 mm3to about 1500 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total volume of about 1350 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

[0093] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragment is obtained by sharp dissection. In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragment is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment is about 10 mm3. In some embodiments, the tumors are 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumors are 1 mm x 1 mm x 1 mm. In some embodiments, the tumors are 2 mm x 2 mm x 2 mm. In some embodiments, the tumors are 3 mm x 3 mm x 3 mm. In some embodiments, the tumors are 4 mm x 4 mm x 4 mm.

[0094] In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic, necrotic, and / or fatty tissues on each piece. In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of necrotic tissue onAtorney Docket No. : 5133- WO each piece. In some embodiments, the tumors are resected in order to minimize the amount of fatty tissue on each piece.

[0095] In some embodiments, the tumor fragmentation is performed in order to maintain the tumor internal structure. In some embodiments, the tumor fragmentation is performed without performing a sawing motion with a scalpel. In some embodiments, the TILs are obtained from tumor digests. In some embodiments, tumor digests were generated by incubation in enzy me media, for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37 °C in 5% CO2 and it then mechanically disrupted again for approximately 1 minute. After being incubated again for 30 minutes at 37 °C in 5% CO2. the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37 °C in 5% CO2. 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, a density gradient separation using Ficoll can be performed to remove these cells.

[0096] In some embodiments, the harvested cell suspension prior to the first expansion step is called a “primary cell population” or a “freshly harvested” cell population.

[0097] In some embodiments, cells can be optionally frozen after sample harvest and stored frozen prior to entry into the expansion described in Step B, which is described in further detail below, as well as exemplified in Figure 1.1. Pleural effusion T-cells and TILs

[0098] In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of the 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 the T-cells or TILs for expansion according to the processes described herein is a pleural effusion derived sample. See, for example, methodsAtorney Docket No. : 5133- WO described in U.S. Patent Publication US 2014 / 0295426, incorporated herein by reference in its entirety for all purposes.

[0099] In some embodiments, any pleural fluid or pleural effusion suspected of and / or containing TILs can be employed. Such a sample may be derived from a primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample may be derived from secondary metastatic cancer cells which originated from another organ, e.g., breast, ovary, colon or prostate. In some embodiments, the sample for use in the expansion methods described herein is a pleural exudate. In some embodiments, the sample for use in the expansion methods described herein is a pleural transudate. Other biological samples may include other serous fluids containing TILs, including, e.g., ascites fluid from the abdomen or pancreatic cyst fluid. Ascites fluid and pleural fluids involve very' similar chemical systems; both the abdomen and lung have mesothelial lines and fluid forms in the pleural space and abdominal spaces in the same matter in malignancies and such fluids in some embodiments contain TILs. In some embodiments, wherein the disclosed methods utilize pleural fluid, the same methods may be performed with similar results using ascites or other cyst fluids containing TILs.

[0100] In some embodiments, the pleural fluid is in unprocessed form, directly as removed from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard blood collection tube, such as an EDTA or Heparin tube, prior to further processing steps. In some embodiments, the unprocessed pleural fluid is placed in a standard CELLSAVE® tube (Veridex) prior to further processing steps. In some embodiments, the sample is placed in the CELLS AVE Wtube immediately after collection from the patient to avoid a decrease in the number of viable TILs. The number of viable TILs can decrease to a significant extent within 24 hours, if left in the untreated pleural fluid, even at 4°C. In some embodiments, the sample is placed in the 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 in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.

[0101] In some embodiments, the pleural fluid sample from the chosen subject may be diluted. In some embodiments, the dilution is L IO pleural fluid to diluent. In other embodiments, the dilution is 1 :9 pleural fluid to diluent. In other embodiments, the dilution is 1 :8 pleural fluid to diluent. In other embodiments, the dilution is 1 :5 pleural fluid to diluent.Atorney Docket No. : 5133- WOIn other embodiments, the dilution is 1 :2 pleural fluid to diluent. In other embodiments, the dilution is 1 : 1 pleural fluid to diluent. In some embodiments, diluents include saline, phosphate buffered saline, another buffer or a physiologically acceptable diluent. In some embodiments, the sample is placed in the CELLSAVE®tube immediately after collection from the patient and dilution to avoid a decrease in the viable TILs, which may occur to a significant extent within 24-48 hours, if left in the untreated pleural fluid, even at 4°C. In some embodiments, the pleural fluid sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient, and dilution. In some embodiments, the pleural fluid sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient, and dilution at 4°C.

[0102] In still other embodiments, pleural fluid samples are concentrated by conventional means prior to further processing steps. In some embodiments, this pre-treatment of the pleural fluid is preferable in circumstances in which the pleural fluid must be cryopreserved for shipment to a laboratory performing the method or for later analysis (e.g., later than 24-48 hours post-collection). In some embodiments, the pleural fluid sample is prepared bycentrifuging the pleural fluid sample after its withdrawal from the subject and resuspending the centrifugate or pellet in buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions, before it is cryopreserved for transport or later analysis and / or processing.

[0103] In some embodiments, pleural fluid samples are concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural fluid sample used in further processing is prepared by filtering the fluid through a filter containing a known and essentially uniform pore size that allows for passage of the pleural fluid through the membrane but retains the tumor cells. In some embodiments, the diameter of the pores in the membrane may be at least 4 pM. In other embodiments the pore diameter may be 5 pM or more, and in other embodiments, any of 6, 7, 8, 9, or 10 pM. After filtration, the cells, including TILs, retained by the membrane may be rinsed off the membrane into a suitable physiologically acceptable buffer. Cells, including TILs, concentrated in this way may then be used in the further processing steps of the method.

[0104] In some embodiments, pleural fluid sample (including, for example, the untreated pleural fluid), diluted pleural fluid, or the resuspended cell pellet, is contacted with a lyticAtorney Docket No. : 5133- WO reagent that differentially lyses non-nucleated red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps in circumstances in which the pleural fluid contains substantial numbers of RBCs. Suitable lysing reagents include a single lytic reagent or a lytic reagent and a quench reagent, or a lytic agent, a quench reagent and a fixation reagent. Suitable lytic systems are marketed commercially and include the BD PHARM LYSE™ system (Becton Dickenson). Other lytic systems include the VERSALYSE™ system, the FACSLYSE™ system (Becton Dickenson), the IMMUNOPREP™ system or Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lytic reagent can vary with the primary requirements being efficient lysis of the red blood cells, and the conservation of the TILs and phenoty pic properties of the TILs in the pleural fluid. In addition to employing a single reagent for lysis, the lytic systems useful in methods described herein can include a second reagent, e.g., one that quenches or retards the effect of the lytic reagent during the remaining steps of the method, e.g., STABILYSE™ reagent (Beckman Coulter, Inc.). A conventional fixation reagent may also be employed depending upon the choice of lytic reagents or the preferred implementation of the method.

[0105] In some embodiments, the pleural fluid sample, unprocessed, diluted or multiply centrifuged or processed as described herein above is cryopreserved at a temperature of about -140 °C prior to being further processed and / or expanded as provided herein.B. STEP B: First Expansion

[0106] In some embodiments, the present methods provide for obtaining young TILs, which are capable of increased replication cycles upon administration to a subject / patient and as such may provide additional therapeutic benefits over older TILs (i.e., TILs which have further undergone more rounds of replication prior to administration to a subject / patient). Features of young TILs have been described in the literature, for example in 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, 25, 53-62: and Tran, et al., J. Immunother.. 2008, 31, 742-751, each of which is incorporated herein by reference.Atorney Docket No. : 5133- WO

[0107] 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), D (diversity). J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs). The present disclosure provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and / or TILs prepared using other methods than those provided herein, including for example, methods other than those embodied in Figure 1. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and / or TILs prepared using methods referred to as process 1C. In some embodiments, the TILs obtained in the first expansion exhibit an increase in the T-cell repertoire diversity. In some embodiments, the increase in diversity is an increase in the immunoglobulin diversity and / or the T-cell receptor diversity. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity' is in the T-cell receptor. 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, there is an increase in the expression of T-cell receptor (TCR) alpha and / or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCRa / (3).

[0108] After dissection or digestion of tumor fragments, for example such as described in Step A of Figure 1, the resulting cells are cultured in serum containing IL -2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum with 6000 lU / mL of IL-2. This primary' cell population is cultured for a period of days, generally from 3 to 14 days, resulting in a bulk TIL population, generally about 1 x 108bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, resulting in a bulk TIL population, generally about 1 * 108bulk TIL cells. In someAtorney Docket No. : 5133- WO embodiments, this primary' cell population is cultured for a period of 10 to 14 days, resulting in a bulk TIL population, generally about 1 x 108bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 x 108bulk TIL cells.

[0109] In some embodiments, expansion of TILs may be performed using an initial bulk TIL expansion step (for example such as those described in Step B of Figure 1, which can include processes referred to as pre-REP) as described below and herein, followed by a second expansion (Step D, including processes referred to as rapid expansion protocol (REP) steps) as described below under Step D and herein, followed by optional cry opreservation, and followed by a second Step D (including processes referred to as restimulation REP steps) as described below and herein. The TILs obtained from this process may be optionally characterized for phenoty pic characteristics and metabolic parameters as described herein.

[0110] In embodiments where TIL cultures are initiated in 24-well plates, for example, using Costar 24-well cell culture cluster, flat bottom (Coming Incorporated, Coming, NY, each well can be seeded with 1 x io6tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 lU / rnL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3.

[0111] In some embodiments, the first expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, CM for Step B consists of RPMI 1640 with GlutaMAX. supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2gas-permeable silicon bottom (for example, G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask was loaded with 10-40 x io6viable tumor digest cells or 5-30 tumor fragments in 10-40 mL of CM with IL-2. Both the G- REX10 and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL- 2 and after day 5, half the media was changed every' 2-3 days.

[0112] In some embodiments, the culture medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or a serum replacement. In some embodiments, the serum-free or defined medium is used toAtorney Docket No. : 5133- WO prevent and / or decrease experimental variation due in part to the lot-to-lot variation of serumcontaining media.

[0113] In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell medium includes, but is not limited to CTS™ OPTMIZER™ T-cell Expansion Basal Medium , CTS™ OPTMIZER™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM). Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0114] In some embodiments, the serum supplement or serum replacement includes, but is not limited to one or more of CTS™ OPTMIZER™T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, MO6+, Ni2+, Rb . Sn2+and Zr4+. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate and / or 2-mercaptoethanol.

[0115] In some embodiments, the CTS™ OPTMIZER™ T-cell Immune Cell Serum Replacement is used with conventional growth media, including but not limited to CTS™ OPTMIZER™ T-cell Expansion Basal Medium, CTS™ OPTMIZER™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (aMEM). Glasgow's Minimal Essential Medium (G-MEM). RPMI growth medium, and Iscove's Modified Dulbecco's Medium.Atorney Docket No. : 5133- WO

[0116] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. 9%, 10%, 11%. 12%. 13%. 14%. 15%. 16%. 17%. 18%. 19%. or 20% by volume of the total serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

[0117] In some embodiments, the serum-free or defined medium is CTS™ OPTMIZER™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OPTMIZER™ may be used. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of IL CTS™ OPTMIZER™ T-cell Expansion Basal Medium and 26 mL CTS™ OPTMIZER™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55mM. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2- mercaptoethanol in the media is 55pM.

[0118] In some embodiments, the defined medium is CTS™ OPTMIZER™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OPTMIZER™ may be used. CTS™ OPTMIZER™ T-cell Expansion SFM is a combination of IL CTS™ OPTMIZER™ T-cell Expansion Basal Medium and 26 mL CTS™ OPTMIZER™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55mM. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L- glutamine. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM isAtorney Docket No. : 5133- WO supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000 lU / mL to about 8000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2- mercaptoethanol, and 2mM of L-glutamine, and further comprises about 3000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 6000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000 lU / mL to about 8000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 3000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 1000 lU / mL to about 6000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 1000 lU / mL to about 8000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 3000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 6000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55pM.Attorney Docket No. : 5133- WO

[0119] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (e.g., GLUTAMAX®) at a concentration of from about 0. 1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM. 2mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (e.g., GLUTAMAX®) at a concentration of about 2 mM.

[0120] In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, 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 media is 55 pM.

[0121] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, are useful in the present disclosure. In that publication, serum-free eukaryotic cell culture media are described. The serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting the growth of cells in serum- free culture. The serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more ingredients selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined medium further comprises L- glutamine, sodium bicarbonate and / or beta-mercaptoethanol. In some embodiments, the defined medium comprises an albumin or an albumin substitute and one or more ingredients selected from group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L- histidine. L-isoleucine. L-methionine, L-Atorney Docket No. : 5133- WO phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moi eties Ag . Al3' , Ba2' , Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, NI2+, Rb+, Sn2+and Zr4+. In some embodiments, the basal cell media is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (aMEM). Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0122] In some embodiments, the concentration of glycine in the defined medium is in the range of from about 5-200 mg / L, the concentration of L- histidine is about 5-250 mg / L, the concentration of L-isoleucine is about 5-300 mg / L, the concentration of L-methionine is about 5-200 mg / L, the concentration of L-phenylalanine is about 5-400 mg / L, the concentration of L-proline is about 1-1000 mg / L, the concentration of L- hydroxyproline is about 1-45 mg / L, the concentration of L-serine is about 1-250 mg / L, the concentration of L- threonine is about 10-500 mg / L, the concentration of L-tryptophan is about 2-110 mg / L, the concentration of L-tyrosine is about 3-175 mg / L, the concentration of L-valine is about 5-500 mg / L, the concentration of thiamine is about 1-20 mg / L, the concentration of reduced glutathione is about 1-20 mg / L, the concentration of L-ascorbic acid-2 -phosphate is about 1- 200 mg / L, the concentration of iron saturated transferrin is about 1-50 mg / L, the concentration of insulin is about 1-100 mg / L, the concentration of sodium selenite is about 0.000001-0.0001 mg / L, and the concentration of albumin (e.g., ALBUMAX® I) is about 5000-50,000 mg / L.

[0123] In some embodiments, the non-trace element moiety ingredients in the defined medium are present in the concentration ranges listed in the column under the heading “Concentration Range in IX Medium” in Table 1 below. In other embodiments, the non-trace element moiety ingredients in the defined medium are present in the final concentrations listed in the column under the heading “A Preferred Embodiment of the IX Medium” 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 moiety ingredients of the type and in the concentrations listed in the column under the heading “A Preferred Embodiment in Supplement” in Table 4 below.Atorney Docket No. : 5133- WOTABLE 1 : Concentrations ofNon-Trace Element Moiety Ingredients

[0124] In some embodiments, the osmolarity' of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L, or about 2.2 g / L sodium bicarbonate. The defined medium can be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA: final concentration of about 100 pM), 2-mercaptoethanol (final concentration of about 100 pM).

[0125] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology. 4(1) 2015 (doi: 10. 1038 / cti.2014.31 ) are useful in the present disclosure. Briefly, RPMI or CTS™ OPTMIZER™ was used as the basal cell medium, and supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.Atorney Docket No. : 5133- WO

[0126] In some embodiments, the cell medium in the first and / or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells. In some embodiments, the cell medium in the first and / or second gas permeable container lacks beta-mercaptoethanol (BME or |3ME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0127] After preparation of the tumor fragments, the resulting cells (z.e., fragments) are cultured in serum containing IL-2 under conditions that favor the grow th of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum (or. in some cases, as outlined herein, in the presence of an APC cell population) with 6000 lU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL population, generally about P I O8bulk TIL cells. In some embodiments, the growth media during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30>< 106lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20xl06lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25xl06lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30x 106HJ / mg for a 1 mg vial. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 xlO6lU / mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7xl06lU / mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6x l06lU / mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in Example 5. In some embodiments, the first expansion culture media comprises about 10,000 lU / mL of IL-2, about 9,000 lU / mL of IL-2, about 8,000 lU / mL of IL-2, about 7,000 lU / mL of IL-2, about 6000 lU / mL of IL-2 or about 5,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 9,000 lU / mL of IL-2 to about 5,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 8,000 lU / mL of IL-2 to about 6,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 7,000 lU / mL of IL-2 to about 6,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 6,000 lU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 lU / mL of IL-2. In someAtorney Docket No. : 5133- WO embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 lU / mL, about 1500 lU / mL, about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000 lU / mL, about 4500 TU / mL, about 5000 lU / mL, about 5500 lU / mL, about 6000 lU / mL, about 6500 lU / mL, about 7000 lU / mL, about 7500 lU / mL, or about 8000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 lU / mL, between 2000 and 3000 lU / mL, between 3000 and 4000 lU / mL, between 4000 and 5000 lU / mL, between 5000 and 6000 lU / mL, between 6000 and 7000 lU / mL, between 7000 and 8000 lU / mL, or about 8000 lU / mL of IL- 2.

[0128] In some embodiments, first expansion culture media comprises about 500 lU / mL of IL-15, about 400 lU / mL of IL-15, about 300 lU / mL of IL-15, about 200 IU / mL of IL-15, about 180 lU / mL of IL-15, about 160 lU / mL of IL-15, about 140 lU / mL of IL-15, about 120 lU / mL of IL-15, or about 100 lU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 500 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 400 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 300 lU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL- 15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL- 15.

[0129] In some embodiments, first expansion culture media comprises about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 5 IU / mL of IL-21 toAtorney Docket No. : 5133- WO about 1 lU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 2 lU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 lU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 lU / mL of IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 lU / mL of IL-21.

[0130] In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, e.g. OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0. 1 ng / mL, about 0.5 ng / mL. about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL. and about 1 pg / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0. 1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0131] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a 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 urelumab, utomilumab. EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, 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 pg / mL and 100 pg / 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 pg / mL and 40 pg / mL.

[0132] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 lU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4- IBB agonist.Atorney Docket No. : 5133- WO

[0133] In some embodiments, the first expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GLUTAMAX®, supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2gas-permeable silicon bottom (for example, G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each flask was loaded with 10-40xl06viable tumor digest cells or 5-30 tumor fragments in 10-40 mL of CM with IL-2. Both the G-REX10 and 24-well plates were incubated in a humidified incubator at 37 °C in 5% CO2 and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5, half the media was changed every 2-3 days. In some embodiments, the CM is the CM1 described in the Examples, see, Example 1. In some embodiments, the first expansion occurs in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium comprises IL-2.

[0134] In some embodiments, the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre-REP) process is shortened to 3-14 days, as discussed in the examples and figures. In some embodiments, the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre- REP) is shortened to 7 to 14 days, as discussed in the Examples and, as well as including for example, an expansion as described in Step B of Figure 1. In some embodiments, the first expansion of Step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, as discussed in, for example, an expansion as described in Step B of Figure 1.

[0135] In some embodiments, the first TIL expansion can proceed 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 expansion can proceed for 1 day to 14 days. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 4 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In someAtorney Docket No. : 5133- WO embodiments, the first TIL expansion can proceed for 7 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 9 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 11 days to 14 days. In some embodiments, the first TIL expansion can proceed for 12 days to 14 days. In some embodiments, the first TIL expansion can proceed for 13 days to 14 days. In some embodiments, the first TIL expansion can proceed for 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 11 days. In some embodiments, the first TIL expansion can proceed for 2 days to 11 days. In some embodiments, the first TIL expansion can proceed for 3 days to 11 days. In some embodiments, the first TIL expansion can proceed for 4 days to 11 days. In some embodiments, the first TIL expansion can proceed for 5 days to 11 days. In some embodiments, the first TIL expansion can proceed for 6 days to 11 days. In some embodiments, the first TIL expansion can proceed for 7 days to 11 days. In some embodiments, the first TIL expansion can proceed for 8 days to 11 days. In some embodiments, the first TIL expansion can proceed for 9 days to 11 days. In some embodiments, the first TIL expansion can proceed for 10 days to 11 days. In some embodiments, the first TIL expansion can proceed for 11 days.

[0136] In some embodiments, a combination of IL-2, IL-7, IL-L5, and / or IL-21 are employed as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL- 15, and / or IL-21 as well as any combinations thereof can be included during the first expansion, including for example during a Step B processes according to Figure 1. as well as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the first expansion. In some embodiments, IL-2, IL-15, and IL-21 as well as any combinations thereof can be included during Step B processes according to Figure 1 and as described herein.

[0137] In some embodiments, the first expansion (including processes referred to as the pre-REP; for example. Step B according to Figure 1) process is shortened to 3 to 14 days, as discussed in the examples and figures. In some embodiments, the first expansion of Step B is shortened to 7 to 14 days. In some embodiments, the first expansion of Step B is shortened to 10 to 14 days. In some embodiments, the first expansion is shortened to 11 days.Atorney Docket No. : 5133- WO

[0138] In some embodiments, the first expansion, for example, Step B according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX-10 or a G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.1. Cytokines and Other Additives

[0139] The expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.

[0140] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is additionally possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is described in U.S. Patent Application Publication No. US 2017 / 0107490 AL the disclosure of which is incorporated by reference herein. 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-2L with the latter finding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein.

[0141] In some embodiments, Step B may also include the addition of OKT-3 antibody or muromonab to the culture media, as described elsewhere herein. In some embodiments, Step B may also include the addition of a 4- IBB agonist to the culture media, as described elsewhere herein. In some embodiments. Step B may also include the addition of an OX-40 agonist to the culture media, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as a thiazolidinedione compound, may be used in the culture media during Step B, as described in U.S. Patent Application Publication No. US 2019 / 0307796 Al, the disclosure of which is incorporated by reference herein.C. STEP C: First Expansion to Second Expansion Transition

[0142] In some cases, the bulk TIL population obtained from the first expansion, including for example the TIL population obtained from for example, Step B as indicated in Figure 1,Atorney Docket No. : 5133- WO can be cryopreserved immediately, using the protocols discussed herein below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which can include expansions sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, in the case where genetically modified TILs will be used in therapy, the first TIL population (sometimes referred to as the bulk TIL population) or the second TIL population (which can in some embodiments include populations referred to as the REP TIL populations) can be subjected to genetic modifications for suitable treatments prior to expansion or after the first expansion and prior to the second expansion.

[0143] In some embodiments, the TILs obtained from the first expansion (for example, from Step B as indicated in Figure 1) are stored until phenotyped for selection. In some embodiments, the TILs obtained from the first expansion (for example, from Step B as indicated in Figure 1) are not stored and proceed directly to the second expansion. In some embodiments, the TILs obtained from the first expansion are not cryopreserved after the first expansion and prior to the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs at 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 from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 3 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 10 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 14 days from when fragmentation occurs.

[0144] In some embodiments, the transition from the first expansion to the second expansion occurs at 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 from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 14 days from when fragmentation occurs. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the transition from the first expansionAtorney Docket No. : 5133- WO to the second expansion occurs 3 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs6 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 1 1 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs7 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 11 days from when fragmentation occurs. In someAtorney Docket No. : 5133- WO embodiments, the transition from the first expansion to the second expansion occurs 10 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days from when fragmentation occurs.

[0145] In some embodiments, the TILs are not stored after the first expansion and prior to the second expansion, and the TILs proceed directly to the second expansion (for example, in some embodiments, there is no storage during the transition from Step B to Step D as shown in Figure 1). In some embodiments, the transition occurs in closed system, as described herein. In some embodiments, the TILs from the first expansion, the second population of TILs. proceeds directly into the second expansion with no transition period.

[0146] In some embodiments, the transition from the first expansion to the second expansion, for example, Step C according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX-10 or a G-REX-100 bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor.D. STEP D: Second Expansion

[0147] In some embodiments, the TIL cell population is expanded in number after harvest and initial bulk processing for example, after Step A and Step B, and the transition referred to as Step C, as indicated in Figure 1. This further expansion is referred to herein as the second expansion, which can include expansion processes generally referred to in the art as a rapid expansion process (REP); as well as processes as indicated in Step D of Figure 1. The second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells, a cytokine source, and an anti-CD3 antibody, in a gas- permeable container.

[0148] In some embodiments, the second expansion or second TIL expansion (which can include expansions sometimes referred to as REP; as well as processes as indicated in Step D of Figure 1) of TIL can be performed using any TIL flasks or containers known by those of skill in the art. In some embodiments, the second TIL expansion can proceed for 7 days, 8 days, 9 days, 10 days, 11 days. 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion can proceed for about 7 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 8 days to about 14 days. In someAtorney Docket No. : 5133- WO embodiments, the second TIL expansion can proceed for about 9 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 10 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[0149] In some embodiments, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including for example, expansions referred to as REP; as well as processes as indicated in Step D of Figure 1). For example, TILs can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin- 15 (IL-15). The non-specific T-cell receptor stimulus can include, for example, an anti-CD3 antibody, such as about 30 ng / mL of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further stimulation of the TILs in vitro by including one or more antigens during the second expansion, including antigenic portions thereof, such as epitope(s). of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 pM MART-1 :26- 35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell grow th factor, such as 300 lU / mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1. TRP-2. tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2. or antigenic portions thereof. TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.Alternatively, the TILs can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the re-stimulation occurs as part of the second expansion. In some embodiments, the second expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0150] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 lU / mL, about 1500 lU / mL.Atorney Docket No. : 5133- WO about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000 lU / mL. about 4500 lU / mL, about 5000 lU / mL, about 5500 lU / mL, about 6000 lU / mL. about 6500 lU / mL. about 7000 lU / mL, about 7500 lU / mL, or about 8000 lU / mL of IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 lU / mL, between 2000 and 3000 lU / mL, between 3000 and 4000 lU / mL, between 4000 and 5000 lU / mL, between 5000 and 6000 lU / mL, between 6000 and 7000 lU / mL, between 7000 and 8000 lU / mL. or between 8000 lU / mL of IL-2.

[0151] In some embodiments, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 pg / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0. 1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0152] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a 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 urelumab, utomilumab. EU-101. a fusion protein, and fragments, derivatives, variants, biosimilars, 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 pg / rnL and 100 pg / 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 pg / mL and 40 pg / mL.

[0153] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 lU / mL and OKT-3Atorney Docket No. : 5133- WO antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4- IBB agonist.

[0154] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL- 15, and / or IL-21 as well as any combinations thereof can be included during the second expansion, including for example during a Step D processes according to Figure 1, as well as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL- 15, and IL-21 as well as any combinations thereof can be included during Step D processes according to Figure 1 and as described herein.

[0155] In some embodiments, the second expansion can be conducted 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 a 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 (APCs; 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).

[0156] In some embodiments, the second expansion culture media comprises about 500 lU / mL of IL-15, about 400 lU / mL of IL-15, about 300 lU / mL of IL-15, about 200 lU / mL of IL-15, about 180 lU / mL of IL-15, about 160 lU / mL of IL-15, about 140 lU / mL of IL-15, about 120 lU / mL of IL-15, or about 100 lU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 500 lU / mL of IL- 15 to about 100 lU / mL of IL- 15. In some embodiments, the second expansion culture media comprises about 400 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 300 lU / mL of IL-15 to about 100 lU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 200 lU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 lU / mL of IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 lU / mL of IL-15.Atorney Docket No. : 5133- WO

[0157] In some embodiments, the second expansion culture media comprises about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 lU / mL of IL-21, about 10 IU / mL of IL- 21. about 5 lU / mL of IL-21, about 4 lU / mL of IL-21, about 3 lU / mL of IL-21, about 2 lU / mL of IL-21, about 1 TU / mL of IL-21 , or about 0.5 lU / mL of IL-21 . In some embodiments, the second expansion culture media comprises about 20 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 15 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 12 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 10 lU / mL of IL-21 to about 0.5 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 5 lU / mL of IL-21 to about 1 lU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL -21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21.

[0158] In some embodiments the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs 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 some embodiments, the ratio of TILs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[0159] In some embodiments, REP and / or the second expansion is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 IU / mL IL-2 in 150 mL media. Media replacement is done (generally 2 / 3 media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber. Alternative grow th chambers include G-REX flasks and gas permeable containers as more fully discussed below;Atorney Docket No. : 5133- WO

[0160] In some embodiments, the second expansion (which can include processes referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second expansion is shortened to 11 days.

[0161] In some embodiments, REP and / or the second expansion may be performed using T- 175 flasks and gas permeable bags as previously descnbed (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas permeable cultureware (G-REX flasks). In some embodiments, the second expansion (including expansions referred to as rapid expansions) is performed in T-175 flasks, and about 1 x 106TILs suspended in 150 mL of media may be added to each T-175 flask. The TILs may be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3. The T-175 flasks may be incubated at 37° C in 5% CO2. Half the media may be exchanged on day 5 using 50 / 50 medium with 3000 IU per mL of IL-2. In some embodiments, on day 7 cells from two T-175 flasks may be combined in a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 mL of TIL suspension. The number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0 x 106cells / mL.

[0162] In some embodiments, the second expansion (which can include expansions referred to as REP, as well as those referred to in Step D of Figure 1) may be performed in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-REX- 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 x 106or 10 x 106TIL may be cultured with PBMCs in 400 mL of 50 / 50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti- CD3 (OKT3). The G-REX-100 flasks may be incubated at 37°C in 5% CO2. On day 5. 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-REX-100 flasks. When TIL are expanded serially in G-REX-100 flasks, on day 7 the TIL in each G-REX-100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3 100 mL aliquots that may be used to seed 3 G-REX- 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask. The G-REX-100 flasks may be incubated at 37° C in 5% CO2Atorney Docket No. : 5133- WO and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G- REX-100 flask. The cells may be harvested on day 14 of culture.

[0163] In some embodiments, the second expansion (including expansions referred to as REP) is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 lU / mL IL-2 in 150 mL media. In some embodiments, media replacement is done until the cells are transferred to an alternative growth chamber. In some embodiments, 2 / 3 of the media is replaced by respiration with fresh media. In some embodiments, alternative growth chambers include G- REX flasks and gas permeable containers as more fully discussed below.

[0164] In some embodiments, the second expansion (including expansions referred to as REP) is performed and further comprises a step wherein TILs are selected for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. Patent Application Publication No. 2016 / 0010058 Al, the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity.

[0165] Optionally, a cell viability assay can be performed after the second expansion (including expansions referred to as the REP expansion), using standard assays known in the art. For example, a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allow s a viability assessment. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to the standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.

[0166] In some embodiments, the second expansion (including expansions referred to as REP) of TIL can be performed using T-175 flasks and gas-permeable bags as previously described (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. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas-permeable G-REX flasks. In some embodiments, the second expansion is performed in T-175 flasks, and about 1 x 106TIL are suspended in about 150 mL of media and this is added to each T-175 flask. The TIL are cultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at a ratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture ofAtorney Docket No. : 5133- WOCM and AIM-V medium (50 / 50 medium), supplemented with 3000 lU / mL of IL-2 and 30 ng / mL of anti-CD3. The T-175 flasks are incubated at 37°C in 5% CO2. In some embodiments, half the media is changed on day 5 using 50 / 50 medium with 3000 lU / mL of IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 lU / mL of IL-2 is added to the 300 mL of TIL suspension. The number of cells in each bag can be counted every day or two and fresh media can be added to keep the cell count between about 0.5 and about 2.0 x 106cells / mL.

[0167] In some embodiments, the second expansion (including expansions referred to as REP) are performed in 500 mL capacity flasks with 100 cm2gas-permeable silicon bottoms (G-REX-100, Wilson Wolf) about 5 x 106or 10 x 106TIL are cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50 / 50 medium, supplemented with 3000 lU / mL of IL-2 and 30 ng / mL of anti-CD3. The G-REX-100 flasks are incubated at 37°C in 5% CO2. In some embodiments, on day 5, 250mL of supernatant is removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellets can then be resuspended with 150 mL of fresh 50 / 50 medium with 3000 IU / mL of IL-2 and added back to the original G-REX-100 flasks. In embodiments where TILs are expanded serially in G-REX-100 flasks, on day 7 the TIL in each G-REX-100 are suspended in the 300 mL of media present in each flask and the cell suspension was divided into three 100 mL aliquots that are used to seed 3 G-REX-100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 lU / mL of IL-2 is added to each flask. The G-REX-100 flasks are incubated at 37°C in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 lU / mL of IL-2 is added to each G-REX-100 flask. The cells are harvested on day 14 of culture.

[0168] 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), D (diversity ), J (joining), and C (constant), determine the binding specificity7and downstream applications of immunoglobulins and T-cell receptors (TCRs). The present disclosure provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity7. In some embodiments, the TILs obtained in the second expansion exhibit an increase in the T-cell repertoire diversity. In some embodiments, the increase in diversity is an increase in the immunoglobulin diversity and / or the T-cellAtorney Docket No. : 5133- WO receptor diversity. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin heavy' chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. 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, there is an increase in the expression of T-cell receptor (TCR) alpha and / or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCRa / p).

[0169] In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells (APCs), as discussed in more detail below.

[0170] In some embodiments, the culture medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and / or decrease experimental variation due in part to the lot-to-lot variation of serumcontaining media.

[0171] In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell medium includes, but is not limited to CTS™ OPTMIZER™ T-cell Expansion Basal Medium , CTS™ OPTMIZER™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LYMPHOONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM). Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0172] In some embodiments, the serum supplement or serum replacement includes, but is not limited to one or more of CTS™ OPTMIZER™T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments.Atorney Docket No. : 5133- WO the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, A13+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, M06+, Ni2+, Rb+, Sn2+ and Zr4+. In some embodiments, the defined medium further comprises L-glutamine. sodium bicarbonate and / or 2- mercaptoethanol.

[0173] In some embodiments, the CTS™ OPTMIZER™ T-cell Immune Cell Serum Replacement is used with conventional growth media, including but not limited to CTS™ OPTMIZER™ T-cell Expansion Basal Medium, CTS™ OPTMIZER™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LYMPHOONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM). Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0174] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. 9%, 10%, 11%. 12%. 13%. 14%. 15%. 16%. 17%. 18%. 19%. or 20% by volume of the total serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

[0175] In some embodiments, the serum-free or defined medium is CTS™ OPTMIZER™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OPTMIZER™ may be used. CTS™ OPTMIZER™ T-cell Expansion SFM is a combination of 1 L CTS™ OPTMIZER™ T-cell Expansion Basal Medium and 26 mL CTS™ OPTMIZER™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™Atorney Docket No. : 5133- WOImmune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55 mM. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2- mercaptoethanol in the media is 55pM.

[0176] In some embodiments, the defined medium is CTS™ OPTMIZER™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OPTMIZER™ may be used. CTS™ OPTMIZER™ T-cell Expansion SFM is a combination of 1 L CTS™ OPTMIZER™ T-cell Expansion Basal Medium and 26 mL CTS™ OPTMIZER™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2- mercaptoethanol at 55mM. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM of 2-mercaptoethanol, and 2 mM of L- glutamine. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). 55 mM of 2-mercaptoethanol. and 2 mM of L-glutamine. and further comprises about 1000 lU / mL to about 8000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM of 2- mercaptoethanol, and 2mM of L-glutamine. and further comprises about 3000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM of 2-mercaptoethanol, and 2 mM of L-glutamine, and further comprises about 6000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM of 2-mercaptoethanol, and further comprises about 1000 lU / mL to about 8000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM of 2- mercaptoethanol, and further comprises about 3000 lU / mL of IL-2. In some embodiments,Atorney Docket No. : 5133- WO the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM of 2- mercaptoethanol, and further comprises about 1000 lU / mL to about 6000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 1000 lU / mL to about 8000 lU / mL of IL-2. In some embodiments, the CTS™ OPTMIZER™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 lU / mL of IL-2. In some embodiments, the CTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 6000 lU / mL of IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55 pM.

[0177] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GLUTAMAX®) at a concentration of from about 0. 1 mM to about 10 mM, 0.5 mM to about 9 rnM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (e.g., GLUTAMAX®) at a concentration of about 2 mM.

[0178] In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 rnM, 40 mM to about 90 rnM, 45 mM to about 85 mM, 50 mM to about 80 rnM, 55 mM to about 75 mM, 60 mM to about 70 mM, 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 media is 55 pM.

[0179] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, are useful in the presentAtorney Docket No. : 5133- WO disclosure. In that publication, serum-free eukaryotic cell culture media are described. The serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting the growth of cells in serum- free culture. The serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more ingredients selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined medium further comprises L- glutamine, sodium bicarbonate and / or beta-mercaptoethanol. In some embodiments, the defined medium comprises an albumin or an albumin substitute and one or more ingredients selected from group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more ingredients selected from the group consisting of glycine, L- histidine. L-isoleucine. L-methionine, L- phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, A13+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+. Br. T, Mn2+, P, Si4+, V5+, M06+, Ni2+, Rb+, Sn2+ and Zr4+. In some embodiments, the basal cell media is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

[0180] In some embodiments, the concentration of glycine in the defined medium is in the range of from about 5-200 mg / L, the concentration of L-histidine is about 5-250 mg / L, the concentration of L-isoleucine is about 5-300 mg / L, the concentration of L-methionine is about 5-200 mg / L, the concentration of L-phenylalanine is about 5-400 mg / L, the concentration of L-proline is about 1-1000 mg / L, the concentration of L-hydroxyproline is about 1-45 mg / L, the concentration of L-serine is about 1-250 mg / L, the concentration of L- threonine is about 1 -500 mg / L, the concentration of L-tryptophan is about 2-110 mg / L, theAtorney Docket No. : 5133- WO concentration of L-tyrosine is about 3-175 mg / L, the concentration of L-valine is about 5-500 mg / L, the concentration of thiamine is about 1 -20 mg / L, the concentration of reduced glutathione is about 1-20 mg / L, the concentration of L-ascorbic acid-2 -phosphate is about 1- 200 mg / L, the concentration of iron saturated transferrin is about 1-50 mg / L, the concentration of insulin is about 1-100 mg / L, the concentration of sodium selenite is about 0.000001-0.0001 mg / L, and the concentration of albumin (e.g., ALBUMAX® I) is about 5000-50,000 mg / L.

[0181] In some embodiments, the non-trace element moiety ingredients in the defined medium are present in the concentration ranges listed in the column under the heading “Concentration Range in I X Medium” in Table 1 . In other embodiments, the non-trace element moiety ingredients in the defined medium are present in the final concentrations listed in the column under the heading “A Preferred Embodiment of the IX Medium” in Table 1. 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 moiety ingredients of the type and in the concentrations listed in the column under the heading “A Preferred Embodiment in Supplement” in Table 1.

[0182] In some embodiments, the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L, or about 2.2 g / L sodium bicarbonate. The defined medium can be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 pM), 2-mercaptoethanol (final concentration of about 100 pM).

[0183] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10. 1038 / cti.2014.31) are useful in the present disclosure. Briefly, RPMI or CTS™ OPTMIZER™ was used as the basal cell medium, and supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0184] In some embodiments, the cell medium in the first and / or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify7the procedures necessary7to expand the number of cells. In some embodiments, the cell medium in the first and / or second gas permeable container lacks beta-mercaptoethanol (BME or (3ME; also known as 2-mercaptoethanol, CAS 60-24-2).Atorney Docket No. : 5133- WO

[0185] In some embodiments, the second expansion, for example, Step D according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX -100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0186] In some embodiments, the step of rapid or second expansion is split into a plurality7of steps to achieve a scaling up of the culture by: (a) performing the rapid or second expansion by culturing TILs in a small scale culture in a first container, e.g., a G-REX-100 MCS container, for a period of about 3 to 7 days, and then (b) effecting the transfer of the TILs in the small scale culture to a second container larger than the first container, e.g., a G- REX-500-MCS container, and culturing the TILs from the small scale culture in a larger scale culture in the second container for a period of about 4 to 7 days.

[0187] In some embodiments, the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out of the culture by: (a) performing the rapid or second expansion by7culturing TILs in a first small scale culture in a first container, e g., a G-REX- 100 MCS container, for a period of about 3 to 7 days, and then (b) effecting the transfer and apportioning of the TILs from the first small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9. 10. 11. 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are equal in size to the first container, wherein in each second container the portion of the TILs from first small scale culture transferred to such second container is cultured in a second small scale culture for a period of about 4 to 7 days.

[0188] In some embodiments, the first small scale TIL culture is apportioned into a plurality of about 2 to 5 subpopulations of TILs.

[0189] In some embodiments, the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out and scaling up of the culture by: (a) performing the rapid or second expansion by culturing TILs in a small scale culture in a first container, e.g., a G- REX-100 MCS container, for a period of about 3 to 7 days, and then (b) effecting the transfer and apportioning of the TILs from the small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are larger in size than the first container, e.g., G-REX-500MCS containers, wherein in each second containerAtorney Docket No. : 5133- WO the portion of the TILs from the small scale culture transferred to such second container is cultured in a larger scale culture for a period of about 4 to 7 days.

[0190] In some embodiments, the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out and scaling up of the culture by: (a) performing the rapid or second expansion by culturing TILs in a small scale culture in a first container, e.g., a G- REX-100 MCS container, for a period of about 5 days, and then (b) effecting the transfer and apportioning of the TILs from the small scale culture into and amongst 2, 3 or 4 second containers that are larger in size than the first container, e g., G-REX-500 MCS containers, wherein in each second container the portion of the TILs from the small scale culture transferred to such second container is cultured in a larger scale culture for a period of about 6 days.

[0191] In some embodiments, upon the splitting of the rapid or second expansion, each second container comprises at least IO8TILs. In some embodiments, upon the splitting of the rapid or second expansion, each second container comprises at least 108TILs, at least 109TILs. or at least IO10TILs. In one exemplary embodiment, each second container comprises at least IO10TILs.

[0192] In some embodiments, the first small scale TIL culture is apportioned into a plurality of subpopulations. In some embodiments, the first small scale TIL culture is apportioned into a plurality' of about 2 to 5 subpopulations. In some embodiments, the first small scale TIL culture is apportioned into a plurality’ of about 2, 3, 4, or 5 subpopulations.

[0193] In some embodiments, after the completion of the rapid or second expansion, the plurality of subpopulations comprises a therapeutically effective amount of TILs. In some embodiments, after the completion of the rapid or second expansion, one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, after the completion of the rapid expansion, each subpopulation of TILs comprises a therapeutically effective amount of TILs.

[0194] In some embodiments, the rapid or second expansion is performed for a period of about 3 to 7 days before being split into a plurality’ of steps. In some embodiments, the splitting of the rapid or second expansion occurs at about day 3, day 4, day 5, day 6, or day 7 after the initiation of the rapid or second expansion.Atorney Docket No. : 5133- WO

[0195] In some embodiments, the splitting of the rapid or second expansion occurs at about day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15. day 16, day 17, or day 18 after the initiation of the first expansion (i.e., pre-REP expansion). In one exemplary embodiment, the splitting of the rapid or second expansion occurs at about day 16 after the initiation of the first expansion.

[0196] In some embodiments, the rapid or second expansion is further performed for a period of about 7 to 11 days after the splitting. In some embodiments, the rapid or second expansion is further performed for a period of about 5 days. 6 days. 7 days. 8 days. 9 days, 10 days, or 11 days after the splitting.

[0197] In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises the same components as the cell culture medium used for the rapid or second expansion after the splitting. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises different components from the cell culture medium used for the rapid or second expansion after the splitting.

[0198] In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, optionally OKT-3 and further optionally APCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, OKT-3, and further optionally APCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2. OKT-3 and APCs.

[0199] In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by supplementing the cell culture medium in the first expansion with fresh culture medium comprising IL-2, optionally OKT-3 and further optionally APCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by supplementing the cell culture medium in the first expansion with fresh culture medium comprising IL-2. OKT-3 and APCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3 and further optionally APCs. In some embodiments, the cell culture medium used for the rapid or second expansion beforeAtorney Docket No. : 5133- WO the splitting is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3 and APCs.

[0200] In some embodiments, the cell culture medium used for the rapid or second expansion after the splitting comprises IL-2, and optionally OKT-3. In some embodiments, the cell culture medium used for the rapid or second expansion after the splitting comprises IL-2, and OKT-3. In some embodiments, the cell culture medium used for the rapid or second expansion after the splitting is generated by replacing the cell culture medium used for the rapid or second expansion before the splitting with fresh culture medium comprising IL-2 and optionally OKT-3. In some embodiments, the cell culture medium used for the rapid or second expansion after the splitting is generated by replacing the cell culture medium used for the rapid or second expansion before the splitting with fresh culture medium comprising IL-2 and OKT-3.

[0201] In some embodiments, the splitting of the rapid expansion occurs in a closed system.

[0202] In some embodiments, the scaling up of the TIL culture during the rapid or second expansion comprises adding fresh cell culture medium to the TIL culture (also referred to as feeding the TILs). In some embodiments, the feeding comprises adding fresh cell culture medium to the TIL culture frequently. In some embodiments, the feeding comprises adding fresh cell culture medium to the TIL culture at a regular interval. In some embodiments, the fresh cell culture medium is supplied to the TILs via a constant flow. In some embodiments, an automated cell expansion system such as Xuri W25 is used for the rapid expansion and feeding.1. Feeder Cells and Antigen Presenting Cells

[0203] In some embodiments, the second expansion procedures described herein (for example including expansion such as those described in Step D from Figure 1, as well as those referred to as REP) require an excess of feeder cells during REP TIL expansion and / or during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

[0204] In general, the allogeneic PBMCs are inactivated, either via irradiation or heat treatment, and used in the REP procedures, as described in the examples, which provides anAtorney Docket No. : 5133- WO exemplary7protocol for evaluating the replication incompetence of irradiate allogeneic PBMCs.

[0205] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion).

[0206] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 lU / mL IL-2.

[0207] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 5-60 ng / mL OKT3 antibody and 1000-6000 lU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 lU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 lU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 25-35 ng / mL OKT3 antibody and 2500-3500 lU / mL IL-2.

[0208] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs 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 some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.Atorney Docket No. : 5133- WO

[0209] In some embodiments, the second expansion procedures described herein require a ratio of about 2.5xl09feeder cells to about lOOxlO6TIL. In other embodiments, the second expansion procedures described herein require a ratio of about 2.5xl09feeder cells to about 50x106TIL. In yet other embodiments, the second expansion procedures described herein require about 2.5xl09feeder cells to about 25xl06TIL.

[0210] In some embodiments, the second expansion procedures described herein require an excess of feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll- Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0211] In general, the allogeneic PBMCs are inactivated, either via irradiation or heat treatment, and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.

[0212] In some embodiments, artificial antigen presenting cells are used in the second expansion as a replacement for, or in combination with. PBMCs.2. Cytokines and Other Additives

[0213] The expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.

[0214] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is additionally possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is described in U.S. Patent Application Publication No. US 2017 / 0107490 AL the disclosure of which is incorporated by reference herein. 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 finding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein.

[0215] In some embodiments, Step D may also include the addition of OKT-3 antibody or muromonab to the culture media, as described elsewhere herein. In some embodiments, Step D may also include the addition of a 4- IBB agonist to the culture media, as describedAtorney Docket No. : 5133- WO elsewhere herein. In some embodiments, Step D may also include the addition of an OX-40 agonist to the culture media, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as athiazolidinedione compound, may be used in the culture media during Step D, as described in U.S. Patent Application Publication No. US 2019 / 0307796 Al, the disclosure of which is incorporated by reference herein.E. STEP E: Harvest TILs

[0216] After the second expansion step, cells can be harvested. In some embodiments the TILs are harvested after one, two, three, four or more expansion steps, for example as provided in Figure 1. In some embodiments the TILs are harvested after two expansion steps, for example as provided in Figure 1.

[0217] TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such know methods can be employed with the present process. In some embodiments, TILs are harvested using an automated system.

[0218] Cell harvesters and / or cell processing systems are commercially available from a variety7of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell based harvester can be employed with the present methods. In some embodiments, the cell harvester and / or cell processing systems is a membrane-based cell harvester. In some embodiments, cell harvesting is via 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 comprising cells through a membrane or filter such as a spinning membrane or spinning filter in a sterile and / or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without pelletization. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid-exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0219] In some embodiments, the harvest, for example, Step E according to Figure 1, is performed from a closed system bioreactor. In some embodiments, a closed system isAtorney Docket No. : 5133- WO employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX-10 or a G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0220] In some embodiments, Step E according to Figure 1, is performed according to the processes described herein. In some embodiments, the closed system is accessed via syringes under sterile conditions in order to maintain the sterility' and closed nature of the system. In some embodiments, a closed system as described in the Examples is employed.

[0221] In some embodiments, TILs are harvested according to the methods described in the Examples. In some embodiments, TILs between days 1 and 11 are harvested using the methods as described in the steps referred herein, such as in the day 1 1 TIL harvest in the Examples. In some embodiments, TILs between days 12 and 24 are harvested using the methods as described in the steps referred herein, such as in the Day 22 TIL harvest in the Examples. In some embodiments, TILs between days 12 and 22 are harvested using the methods as described in the steps referred herein, such as in the Day 22 TIL harvest in the Examples.F. STEP F: Final Formulation and Transfer to Infusion Container

[0222] After Steps A through E as provided in an exemplary' order in Figure 1 and as outlined in detailed above and herein are complete, cells are transferred to a container for use in administration to a patient, such as an infusion bag or sterile vial. In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, they are transferred to a container for use in administration to a patient.

[0223] In some embodiments, TILs expanded using APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, the T-cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.Atorney Docket No. : 5133- WOIII. Methods of Treating Cancer Using TIL, PD-1 / PD-L1 Inhibitor and LAG-3 Inhibitor1. Combinations with PD-1 and PD-L1 Inhibitors

[0224] In some embodiments, the TIL therapy provided to patients with cancer may include treatment with therapeutic populations of TILs alone or may include a combination treatment including TILs and one or more PD-1 and / or PD-L1 inhibitors. In some embodiments, the TIL therapy provided to patients with epithelial cancer may include treatment with therapeutic populations of TILs alone or may include a combination treatment including TILs and one or more PD-1 and / or PD-L1 inhibitors. In some embodiments, the TIL therapy provided to patients with mesothelioma cancer may include treatment with therapeutic populations of TILs alone or may include a combination treatment including TILs and one or more PD-1 and / or PD-L1 inhibitors. In some embodiments, the TIL therapy provided to patients with endothelial cancer may include treatment with therapeutic populations of TILs alone or may include a combination treatment including TILs and one or more PD-1 and / or PD-L1 inhibitors.

[0225] Programmed death 1 (PD-1) is a 288-amino acid transmembrane immunocheckpoint receptor protein expressed by T cells, B cells, natural killer (NK) T cells, activated monocytes, and dendritic cells. PD-1, which is also known as CD279, belongs to the CD28 family, and in humans is encoded by the Pdcdl gene on chromosome 2. PD-1 consists of one immunoglobulin (Ig) superfamily domain, a transmembrane region, and an intracellular domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor ty rosine-based switch motif (ITSM). PD-1 and its ligands (PD-L1 and PD- L2) are known to play a key role in immune tolerance, as described in Keir, el al., Annu. Rev. Immunol. 2008, 26, 677-704. PD-1 provides inhibitory signals that negatively regulate T cell immune responses. PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7- DC or CD273) are expressed on tumor cells and stromal cells, which may be encountered by activated T cells expressing PD-1, leading to 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 by use of a PD-1 inhibitor, a PD-L1 inhibitor, and / or a PD-L2 inhibitor can overcome immune resistance, as demonstrated in recent clinical studies, such as that described in Topalian, et al., N. Eng. J. Med. 2012, 366. 2443-54. PD-L1 is expressed on many tumor cell lines, while PD-L2 isAtorney Docket No. : 5133- WO expressed mostly on dendritic cells and a few tumor lines. In addition to T cells (which inducibly express PD-1 after activation), PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes, and dendritic cells.

[0226] In some embodiments, the TILs produced as described herein can be administered in combination with one or more PD-1 inhibitors for treating cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more PD-1 inhibitors for treating cancer in a patient or subject, further combining with one or more LAG-3 inhibitors. In some embodiments, the TILs produced as described herein can be administered in combination with one or more PD-L1 inhibitors for treating cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more PD-L1 inhibitors for treating cancer in a patient or subject, further combining with one or more LAG-3 inhibitors. In some embodiments, the TILs produced as described herein can be administered in combination with one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating cancer in a patient or subject, further combining with one or more LAG-3 inhibitors.

[0227] In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof. In some embodiments, the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colonAtorney Docket No. : 5133- WO cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC). hypopharynx cancer, larynx cancer, nasopharynx cancer, oropharynx cancer, and pharynx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the patient or subject was previously treated with one or more immune checkpoint inhibitors.

[0228] In some embodiments, the cancer is melanoma. In some embodiments, the cancer is unresectable or metastatic melanoma. In embodiments, the subject was previously treated with a PD-1 blocking antibody, and if BRAF V600 mutation positive, a BRAF inhibitor with or without a MEK inhibitor. In some embodiments, the cancer is mucosal melanoma. In some embodiments, the cancer is uveal melanoma. In some embodiments, the cancer is choroidal melanoma. In some embodiments, the cancer is ciliary body melanoma. In some embodiments, the cancer is iris melanoma.

[0229] In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is HNSCC. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma. and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC).Atorney Docket No. : 5133- WO hypopharynx cancer, larynx cancer, nasopharynx cancer, oropharynx cancer, and pharynx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer. In embodiments, primary mediastinal large B-cell lymphoma (PMBCL) is treated. In some embodiments, classical Hodgkin lymphoma is treated. In some embodiments, Recurrent or metastatic squamous cell carcinoma of the head and neck is treated. In some embodiments, locally advanced or metastatic urothelial carcinoma is treated. In some embodiments, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer is treated. In some embodiments, hepatocellular carcinoma is treated. In some embodiments, esophageal squamous cell carcinoma is treated.

[0230] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0231] In some embodiments, TILs and a PD-1 inhibitor are administered as a combination therapy or co-therapy for the treatment of melanoma.

[0232] In some embodiments, the melanoma has undergone no prior therapy. In some embodiments, a PD-1 inhibitor is administered as a front-line therapy or initial therapy. In some embodiments, a PD-1 inhibitor is administered as a front-line therapy or initial therapy in combination with the TILs as described herein.

[0233] In some embodiments, the PD-1 inhibitor may be any PD-1 inhibitor or PD-1 blocker known in the art. In particular, it is one of the PD-1 inhibitors or blockers described in more detail in the following paragraphs. The terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein in reference to PD-1 inhibitors. For avoidance of doubt, references herein to a PD-1 inhibitor that is an antibody may refer to a compound or antigenbinding fragments, variants, conjugates, or biosimilars thereof. For avoidance of doubt,Atorney Docket No. : 5133- WO references herein to a PD-1 inhibitor may also refer to a small molecule compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cociystal, or prodrug thereof.

[0234] In some embodiments, the PD-1 inhibitor is an antibody (z.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 some embodiments, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competes for binding with PD-1, and / or binds to an epitope on PD-1. In some embodiments, the antibody competes for binding with PD-1, and / or binds to an epitope on PD-1.

[0235] In some embodiments, the PD-1 inhibitor is one that binds human PD-1 with a KD of about 100 pM or lower, binds human PD-1 with a KD of about 90 pM or lower, binds human PD-1 with a KD of about 80 pM or lower, binds human PD-1 with a KD of about 70 pM or low er, binds human PD-1 w ith a KD of about 60 pM or low er, binds human PD-1 w ith a KD of about 50 pM or lower, binds human PD-1 with a KD of about 40 pM or lower, binds human PD-1 with a KD of about 30 pM or lower, binds human PD-1 with a KD of about 20 pM or lower, binds human PD-1 with a KD of about 10 pM or lower, or binds human PD-1 with a KD of about 1 pM or low er.

[0236] In some embodiments, the PD-1 inhibitor is one that binds to human PD-1 with a kassoe of about 7.5 x io51 / M s or faster, binds to human PD-1 with a kassoc of about 7.5 x io51 / M s or faster, binds to human PD-1 with a kassoc of about 8 x io51 / M s or faster, binds to human PD-1 with a kassoc of about 8.5 x io51 / M s or faster, binds to human PD-1 with a kassoc of about 9 x 1051 / M s or faster, binds to human PD-1 with a kassoc of about 9.5 x 1051 / M s or faster, or binds to human PD-1 with a kassoc of about 1 x io61 / M s or faster.

[0237] In some embodiments, the PD-1 inhibitor is one that binds to human PD-1 with a kdissoc of about 2 x 10'51 / s or slower, binds to human PD-1 with a kdissoc of about 2.1 x 10'51 / s or slower , binds to human PD-1 with a kdissoc of about 2.2 x 10’51 / s or slower, binds to human PD-1 with a kdissoc of about 2.3 x 10-5 1 / s or slower, binds to human PD-1 with a kdissoc of about 2.4 x 10'51 / s or slower, binds to human PD-1 with a kdissoc of about 2.5 x 10'51 / s or slower, binds to human PD-1 with a kdissoc of about 2.6 x 10'51 / s or slower or binds to human PD-1 with a kdissoc of about 2.7 x IO’31 / s or slow er, binds to human PD-1 with a kdissoc of about 2.8 x 10’51 / s or slower, binds to human PD-1 with a kdissoc of about 2.9 x 10’51 / s or slower, or binds to human PD-1 with a kdissoc of about 3 x 10’51 / s or slower.Atorney Docket No. : 5133- WO

[0238] In some embodiments, the PD-1 inhibitor is one that blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 10 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 9 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 8 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 7 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 6 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 5 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower, blocks or inhibits binding of human PD- L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or low er, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or low er, or blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 w ith an IC50 of about 1 nM or low er.

[0239] In some embodiments, the PD-1 inhibitor is nivolumab (commercially available as OPDIVO from Bristol-Myers Squibb Co.), or biosimilars, antigen-binding fragments, conjugates, or variants thereof. Nivolumab is a fully human IgG4 antibody blocking the PD-1 receptor. In some embodiments, the anti-PD-1 antibody is an immunoglobulin G4 kappa, anti-(human CD274) antibody. Nivolumab is assigned Chemical Abstracts Service (CAS) registry' number 946414-94-4 and is 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 No. WO 2006 / 121168, the disclosures of which are incorporated by reference herein. The 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 by reference herein. Nivolumab has intra-heavy chain disulfide linkages at 22-96,140-196, 254-314, 360-418, 22"-96", 140"-196", 254"-314", and 360"-418"; intra-hght chain disulfide linkages at 23'-88', 134'- 194', 23"'-88"', and 134"'-194'"; inter-heav -light chain disulfide linkages at 127-214', 127"-214'", inter-heavy-heavy chain disulfide linkages at 219-219" and 222-222"; and N- glycosylation sites (H CH2 84.4) at 290. 290".Atorney Docket No. : 5133- WO

[0240] In some embodiments, the PD-1 inhibitor is an anti-PD-1 biosimilar monoclonal antibody approved by drug regulatory authorities with reference to nivolumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity7, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-1 antibody authorized or submitted for authorization, wherein the anti-PD-1 antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab. The anti-PD-1 antibody may be authorized by a drug regulatory' authority' such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab.

[0241] In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and the nivolumab is administered at a dose of about 0.5 mg / kg to about 10 mg / kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and the nivolumab is administered at a dose of about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg. about 9 mg / kg, about 9.5 mg / kg, or about 10 mg / kg. In some embodiments, the nivolumab administration is begun 1. 2, 3, 4. or 5 days post IL-2 administration. In some embodiments, the nivolumab administration is begun 1 , 2, or 3 daysAtorney Docket No. : 5133- WO post IL-2 administration. In some embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (z'.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the nivolumab can also be administered 1, 2. or 3 weeks preresection (z'.e., before obtaining a tumor sample from the subject or patient).

[0242] In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and the nivolumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and the nivolumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg. about 280 mg, about 300 mg, about 320 mg. about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, the nivolumab administration is begun 1, 2, 3, 4, or 5 days post IL-2 administration. In some embodiments, the nivolumab administration is begun 1, 2, or 3 days post IL-2 administration. In some embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the nivolumab can also be administered 1, 2, or 3 weeks preresection (z. e. , before obtaining a tumor sample from the subj ect or patient).

[0243] In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, and the nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the nivolumab administration is begun 1, 2. 3, 4, or 5 days post IL-2 administration. In some embodiments, the nivolumab administration is begun 1, 2, or 3 days post IL-2 administration. In some embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (z'.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the nivolumab can also be administered 1, 2, or 3 weeks pre-resection (z.e.. before obtaining a tumor sample from the subject or patient).

[0244] In some embodiments, the nivolumab is administered at about 240 mg every 2 weeks. In some embodiments, the nivolumab is administered at about 480 mg every 4 weeks. In some embodiments, the nivolumab is administered at about 240 mg every 2 weeks or 480 mg every 4 weeks. In some embodiments, the nivolumab is administered at about 1 mg / kg followed by ipilimumab 3 mg / kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks or 480 mg every 4 weeks.

[0245] In some embodiments, the nivolumab administration is begun 1. 2, 3, 4. or 5 days post IL-2 administration. In some embodiments, the nivolumab administration is begun 1, 2,Atorney Docket No. : 5133- WO or 3 days post IL-2 administration. In some embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (z.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the nivolumab can also be administered 1. 2, or 3 weeks pre-resection (z.e., before obtaining a tumor sample from the subject or patient).

[0246] In some embodiments, the nivolumab is administered at about 3 mg / kg every 2 weeks along with ipilimumab at about 1 mg / kg every 6 weeks. In some embodiments, the nivolumab is administered at about 360 mg every73 weeks with ipilimumab 1 mg / kg every76 weeks and 2 cycles of platinum-doublet chemotherapy. In some embodiments, the nivolumab administration is begun 1, 2. 3, 4, or 5 days post IL-2 administration. In some embodiments, the nivolumab administration is begun 1 , 2, or 3 days post IL-2 administration. In some embodiments, the nivolumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (z.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the nivolumab can also be administered 1. 2, or 3 weeks pre-resection (i.e.. before obtaining a tumor sample from the subject or patient). In some embodiments, the nivolumab is administered at about 360 mg every 3 weeks with ipilimumab 1 mg / kg every76 weeks.

[0247] In some embodiments, the nivolumab is administered at about 3 mg / kg followed by ipilimumab at about 1 mg / kg on the same day every73 weeks for 4 doses, then 240 mg every72 weeks. In some embodiments, the nivolumab is administered at about 3 mg / kg followed by ipilimumab at about 1 mg / kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks or 480 mg every74 weeks.

[0248] In other embodiments, the PD-1 inhibitor comprises pembrolizumab (commercially available as KEYTRUDA from Merck & Co., Inc., Kenilworth, NJ, USA), or antigenbinding fragments, conjugates, or variants thereof. Pembrolizumab is assigned CAS registry7number 1374853-91-4 and is also known as lambrolizumab, MK-3475, and SCH-900475. Pembrolizumab has an immunoglobulin G4, anti-(human protein PDCD 1 (programmed cell death 1)) (human-Mus musculus monoclonal heavy chain), disulfide with human-Mus musculus monoclonal light chain, dimer structure. The structure of pembrolizumab may also be described as immunoglobulin G4, anti-(human programmed cell death 1); humanized mouse monoclonal [228-L-proline(H10-S>P)]y4 heavy chain (134-218')-disulfide with humanized mouse monoclonal K light chain dimer (226-226":229-229")-bisdisulfide. The properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO 2008 / 156712 Al, U.S. Patent No. 8,354,509 and U.S. PatentAtorney Docket No. : 5133- WOApplication Publication Nos. US 2010 / 0266617 Al, US 2013 / 0108651 Al, and US 2013 / 0109843 A2, the disclosures of which are incorporated herein by reference. The clinical safety and efficacy of pembrolizumab in various forms of cancer is described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert, et al., lancet, 2014, 384, 1109-17; and Thomas, et al., Exp. Opin. Biol. Ther., 2014, 14, 1061-1064. Pembrolizumab includes the following disulfide bridges: 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 an IgG4 / kappa isotype with a stabilizing S228P mutation in the Fc region; insertion of this mutation in the IgG4 hinge region prevents the formation of half molecules ty pically observed for IgG4 antibodies. Pembrolizumab is heterogeneously glycosylated at Asn297 within the Fc domain of each heavy chain, yielding a molecular weight of approximately 149 kDa for the intact antibody. The dominant glycoform of pembrolizumab is the fucosylated agalacto diantennary glycan form (G0F).

[0249] In some embodiments, the PD-1 inhibitor is an anti-PD-1 biosimilar monoclonal antibody approved by drug regulatory authorities with reference to pembrolizumab. In some embodiments, the biosimilar comprises an anti-PD-1 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity7, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is pembrolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-1 antibody authorized or submitted for authorization, wherein the anti-PD- 1 antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is pembrolizumab. The anti-PD-1 antibody may be authorized by a drug regulatory7authority7such as the U.S. FDA and / or the European Union’s EMA. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product,Atorney Docket No. : 5133- WO wherein the reference medicinal product or reference biological product is pembrolizumab. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is pembrolizumab.

[0250] In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the pembrolizumab is administered at a dose of about 0.5 mg / kg to about 10 mg / kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the pembrolizumab is administered at a dose of about 0.5 mg / kg. about 1 mg / kg. about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg. about 9 mg / kg, about 9.5 mg / kg, or about 10 mg / kg. In some embodiments, the pembrolizumab administration is begun 1, 2. 3, 4, or 5 days post IL-2 administration. In some embodiments, the pembrolizumab administration is begun 1, 2, or 3 days post IL-2 administration. In some embodiments, the pembrolizumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (z.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the pembrolizumab can also be administered 1, 2, or 3 weeks pre-resection (i.e.. before obtaining a tumor sample from the subject or patient).

[0251] In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein the pembrolizumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, and the nivolumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg. about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg. In some embodiments, the pembrolizumab administration is begun 1, 2, 3, 4, or 5 days post IL-2 administration. In some embodiments, the pembrolizumab administration is begun 1, 2, or 3 days post IL-2 administration. In some embodiments, the pembrolizumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (i.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the pembrolizumab can also be administered 1, 2, or 3 weeks pre-resection (i.e.. before obtaining a tumor sample from the subject or patient).Atorney Docket No. : 5133- WO

[0252] In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein the pembrolizumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the pembrolizumab administration is begun 1, 2, 3, 4, or 5 days post IL-2 administration. In some embodiments, the pembrolizumab administration is begun 1, 2, or 3 days post IL-2 administration. In some embodiments, the pembrolizumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (z.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the pembrolizumab can also be administered 1, 2, or 3 weeks pre-resection (i.e., before obtaining a tumor sample from the subject or patient).

[0253] In some embodiments, if the patient or subject is an adult, z.e., treatment of adult indications, and additional dosing regimen of 400 mg every' 6 weeks can be employed. In some embodiments, the pembrolizumab administration is begun 1, 2, 3, 4, or 5 days post IL-2 administration. In some embodiments, the pembrolizumab administration is begun 1. 2, or 3 days post IL-2 administration. In some embodiments, the pembrolizumab can also be administered 1, 2, 3, 4 or 5 weeks pre-resection (z.e., before obtaining a tumor sample from the subject or patient). In some embodiments, the pembrolizumab can also be administered 1, 2, or 3 weeks pre-resection (z.e., before obtaining a tumor sample from the subject or patient).

[0254] In some embodiments, the PD-1 inhibitor is a commercially-available anti-PD-1 monoclonal antibody, such as anti-m-PD-1 clones J43 (Cat # BE0033-2) and RMP1-14 (Cat # BE0146) (Bio X Cell, Inc., West Lebanon, NH, USA). A number of commercially- available anti-PD-1 antibodies are known to one of ordinary' skill in the art.

[0255] In some embodiments, the PD-1 inhibitor is an antibody disclosed in U.S. Patent No. 8,354,509 or U.S. Patent Application Publication Nos. 2010 / 0266617 Al, 2013 / 0108651 Al, 2013 / 0109843 A2, the disclosures of which are incorporated by reference herein. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody described in U.S. Patent Nos. 8,287,856, 8,580,247, and 8,168,757 and U.S. Patent Application Publication Nos.2009 / 0028857 Al, 2010 / 0285013 Al, 2013 / 0022600 Al, and 2011 / 0008369 Al, the teachings of which are hereby incorporated by reference. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in U.S. Patent No. 8,735.553 Bl, the disclosure of which is incorporated herein by reference. In some embodiments, the PD-1 inhibitor is pidilizumab, also known as CT-011, which is described in U.S. Patent No. 8,686,1 19, the disclosure of which is incorporated by reference herein.Atorney Docket No. : 5133- WO

[0256] In some embodiments, the PD-1 inhibitor may be a small molecule or a peptide, or a peptide derivative, such as those described in U.S. Patent Nos. 8,907,053; 9,096,642; and 9,044,442 and U.S. Patent Application Publication No. US 2015 / 0087581; 1.2.4-oxadi azole compounds and derivatives such as those described in U.S. Patent Application Publication No. 2015 / 0073024; cyclic peptidomimetic compounds and derivatives such as those described in U.S. Patent Application Publication No. US 2015 / 0073042; cyclic compounds and derivatives such as those described in U.S. Patent Application Publication No. US 2015 / 0125491; 1,3,4-oxadiazole and 1,3,4-thiadiazole compounds and derivatives such as 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 a macrocyclic peptide-based compounds and derivatives such as those described in U.S. Patent Application Publication No. US 2014 / 0294898; the disclosures of each of which are hereby incorporated by reference in their entireties. In some embodiments, the PD-1 inhibitor is cemiplimab, which is commercially available from Regeneron, Inc.

[0257] In some embodiments, TILs and a PD-L1 inhibitor or a PD-L2 inhibitor are administered as a combination therapy or co-therapy for the treatment of melanoma.

[0258] In some embodiments, the melanoma has undergone no prior therapy. In some embodiments, a PD-L1 inhibitor or a PD-L2 inhibitor is administered as a front-line therapy or initial therapy. In some embodiments, a PD-L1 inhibitor or a PD-L2 inhibitor is administered as a front-line therapy or initial therapy in combination with the TILs as described herein.

[0259] In some embodiments, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD- L2 inhibitors, antagonist, or blockers described in more detail in the following paragraphs. The terms ‘'inhibitor,” '‘antagonist,” and “blocker” are used interchangeably herein in reference to PD-L1 and PD-L2 inhibitors. For avoidance of doubt, references herein to a PD- L1 or PD-L2 inhibitor that is an antibody may refer to a compound or antigen-binding fragments, variants, conjugates, or biosimilars thereof. For avoidance of doubt, references herein to a PD-L1 or PD-L2 inhibitor may refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocry stal, or prodrug thereof.Atorney Docket No. : 5133- WO

[0260] In some embodiments, the compositions, processes and methods described herein include a PD-L1 or PD-L2 inhibitor. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In some embodiments, the PD-L1 or PD-L2 inhibitor is an antibody ( / .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 some embodiments, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competes for binding with PD-L1 or PD-L2, and / or binds to an epitope on PD-L1 or PD-L2. In some embodiments, the antibody competes for binding with PD-L1 or PD-L2, and / or binds to an epitope on PD-L1 or PD-L2.

[0261] In some embodiments, the PD-L1 inhibitors provided herein are selective for PD- Ll, in that the compounds bind or interact with PD-L1 at substantially lower concentrations than they bind or interact with other receptors, including the PD-L2 receptor. In certain embodiments, the compounds bind to the PD-L1 receptor at a binding constant that is at least about a 2-fold higher concentration, about a 3-fold higher concentration, about a 5-fold higher concentration, about a 10-fold higher concentration, about a 20-fold higher concentration, about a 30-fold higher concentration, about a 50-fold higher concentration, about a 100-fold higher concentration, about a 200-fold higher concentration, about a 300- fold higher concentration, or about a 500-fold higher concentration than to the PD-L2 receptor.

[0262] In some embodiments, the PD-L2 inhibitors provided herein are selective for PD- L2, in that the compounds bind or interact with PD-L2 at substantially lower concentrations than they bind or interact with other receptors, including the PD-L1 receptor. In certain embodiments, the compounds bind to the PD-L2 receptor at a binding constant that is at least about a 2-fold higher concentration, about a 3-fold higher concentration, about a 5-fold higher concentration, about a 10-fold higher concentration, about a 20-fold higher concentration, about a 30-fold higher concentration, about a 50-fold higher concentration, about a 100-fold higher concentration, about a 200-fold higher concentration, about a 300- fold higher concentration, or about a 500-fold higher concentration than to the PD-L1 receptor.

[0263] Without being bound by any theory, it is believed that tumor cells express PD-L1, and that T cells express PD-1. However, PD-L1 expression by tumor cells is not required forAtorney Docket No. : 5133- WO efficacy of PD-1 or PD-L1 inhibitors or blockers. In some embodiments, the tumor cells express PD-L1. In other embodiments, the tumor cells do not express PD-L1. In some embodiments, the methods can include a combination of a PD-1 and a PD-L1 antibody, such as those described herein, in combination with a TIL. The administration of a combination of a PD-1 and a PD-L1 antibody and a TIL may be simultaneous or sequential.

[0264] In some embodiments, the PD-L1 and / or PD-L2 inhibitor is one that binds human PD-L1 and / or PD-L2 with a KD of about 100 pM or lower, binds human PD-L1 and / or PD- L2 with a KD of about 90 pM or lower, binds human PD-L1 and / or PD-L2 with a KD of about 80 pM or lower, binds human PD-L1 and / or PD-L2 with a KD of about 70 pM or lower, binds human PD-L1 and / or PD-L2 with a KD of about 60 pM or lower, a KD of about 50 pM or lower, binds human PD-L1 and / or PD-L2 with a KD of about 40 pM or lower, or binds human PD-L1 and / or PD-L2 with a KD of about 30 pM or lower,

[0265] In some embodiments, the PD-L1 and / or PD-L2 inhibitor is one that binds to human PD-L1 and / or PD-L2 with a kassoc of about 7.5 x 1051 / M s or faster, binds to human PD-L1 and / or PD-L2 with a kassoc of about 8 x 1051 / M s or faster, binds to human PD-LI and / or PD-L2 with a kassoc of about 8.5 x IO31 / M s or faster, binds to human PD-LI and / or PD-L2 with a kassoc of about 9 x 1051 / M s or faster, binds to human PD-LI and / or PD-L2 with a kassoc of about 9.5 x 1051 / M s and / or faster, or binds to human PD-LI and / or PD-L2 with a kassoc of about 1 x io61 / M s or faster.

[0266] In some embodiments, the PD-LI and / or PD-L2 inhibitor is one that binds to human PD-LI or PD-L2 with a kdissoc of about 2 x 10'51 / s or slower, binds to human PD-1 with a kdissoc of about 2.1 x 10'51 / s or slower , binds to human PD-1 with a kdissoc of about 2.2 x 10’31 / s or slow er, binds to human PD-1 with a kdissoc of about 2.3 x 10'51 / s or slower, binds to human PD-1 with a kdissoc of about 2.4 x 10-5 1 / s or slower, binds to human PD-1 with a kdissoc of about 2.5 x 10’51 / s or slower, binds to human PD-1 with a kdissoc of about 2.6 x 10’51 / s or slower, binds to human PD-LI or PD-L2 with a kdissoc of about 2.7 x 10'51 / s or slow er, or binds to human PD-LI or PD-L2 with a kdissoc of about 3 x 10'51 / s or slower.

[0267] In some embodiments, the PD-LI and / or PD-L2 inhibitor is one that blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 10 nM or lower; blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 9 nM or lower; blocks or inhibits binding of human PD-LI or human PD-L2 to human PD-1 with an IC50 of about 8 nM or lower; blocks or inhibits binding ofAtorney Docket No. : 5133- WO human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 7 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 6 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an TC5O of about 5 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 4 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 3 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 2 nM or lower; or blocks human PD-1, or blocks binding of human PD-L1 or human PD-L2 to human PD-1 with an IC50 of about 1 nM or lower.

[0268] In some embodiments, the PD-L1 inhibitor is durvalumab. also known as MEDI4736 (which is commercially available from Medimmune, LLC, Gaithersburg, Maryland, a subsidiary of AstraZeneca pic.), or antigen-binding fragments, conjugates, or variants thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013 / 0034559, the disclosures of which are incorporated by reference herein. The clinical efficacy of durvalumab has been described in Page, et al., Ann. Rev. Med., 2014, 65, 185-202; Brahmer, et al.. J. Clin. Oncol. 2014. 32. 5s (supplement, abstract 8021); and McDermott, et al., Cancer Treatment Rev., 2014. 40, 1056-64. The preparation and properties of durvalumab are described in U.S. Patent No. 8,779,108, the disclosure of which is incorporated by reference herein. The durvalumab monoclonal antibody includes disulfide linkages at 22-96, 22"-96", 23'-89', 23'"-89"'. 135'-195', 135'"-195"', 148-204, 148"-204", 215'-224, 215"'-224", 230-230", 233-233", 265-325, 265"-325", 371-429. and 371"-429'; and N-glycosylation sites at Asn-301 and Asn-301".

[0269] In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll biosimilar monoclonal antibody approved by drug regulatory authorities with reference to durvalumab. In some embodiments, the biosimilar comprises an anti-PD-Ll antibody comprising an amino acid sequence which has at least 97% sequence identity', e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is durvalumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation,Atorney Docket No. : 5133- WO oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD- L1 antibody authorized or submitted for authorization, wherein the anti-PD-Ll antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is durvalumab. The anti-PD-Ll antibody may be authorized by a drug regulator ' authority' such as the U.S. FDA and / or the European Union’s EMA. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is durvalumab. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is durvalumab.

[0270] In some embodiments, the PD-L1 inhibitor is avelumab, also known as MSB0010718C (commercially available from Merck KGaA / EMD Serono), or antigenbinding fragments, conjugates, or variants thereof. The preparation and properties of avelumab are described in U.S. Patent Application Publication No. US 2014 / 0341917 Al, the disclosure of which is specifically incorporated by reference herein. Avelumab has intraheavy chain disulfide linkages (C23-C104) at 22-96, 147-203, 264-324, 370-428, 22"-96", 147"-203", 264"-324", and 370"-428"; intra-light chain disulfide linkages (C23-C104) at 22'- 90'. 138'-197', 22"'-90'", and 138"'-197"'; intra-heavy-light chain disulfide linkages (h 5-CL 126) at 223-215' and 223"-215'"; intra-heavy -heavy chain disulfide linkages (h 11, h 14) at 229-229" and 232-232"; N-glycosylation sites (H CH2 N84.4) at 300, 300"; fucosylated complex bi-antennary CHO-ty pe glycans; and H CHS K2 C-terminal lysine clipping at 450 and 450'.

[0271] In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll biosimilar monoclonal antibody approved by drug regulatory authorities with reference to avelumab. In some embodiments, the biosimilar comprises an anti-PD-Ll antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to theAtorney Docket No. : 5133- WO reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is avelumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation. Tn some embodiments, the biosimilar is an anti-PD-Ll antibody authorized or submitted for authorization, wherein the anti-PD-Ll antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is avelumab. The anti-PD-Ll antibody may be authorized by a drug regulatory authority such as the U.S. FDA and / or the European Union’s EMA. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is avelumab. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is avelumab.

[0272] In some embodiments, the PD-L1 inhibitor is atezolizumab. also known as MPDL3280A or RG7446 (commercially available as TECENTRIQ® from Genentech, Inc., a subsidiary of Roche Holding AG, Basel, Switzerland), or antigen-binding fragments, conjugates, or variants thereof. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8.217,149, the disclosure of which is specifically incorporated by reference herein. In some embodiments, the PD-L1 inhibitor is an antibody disclosed in U.S. Patent Application Publication Nos. 2010 / 0203056 Al, 2013 / 0045200 Al, 2013 / 0045201 Al, 2013 / 0045202 Al, or 2014 / 0065135 Al, the disclosures of which are specifically incorporated by reference herein. The preparation and properties of atezolizumab are described in U.S. Patent No. 8,217,149, the disclosure of which is incorporated by reference herein. Atezolizumab has intra-heavy chain disulfide linkages (C23-C104) at 22-96, 145-201, 262-322, 368-426, 22"-96", 145"-201", 262"-322", and 368"-426"; intra-light chain disulfide linkages (C23-C104) at 23'-88', 134'-194', 23"'-88"', and 134"'-194'"; intra-heavy- light chain disulfide linkages (h 5-CL 126) at 221-214' and 221"-214"'; intra-heavy -heavyAtorney Docket No. : 5133- WO chain disulfide linkages (h 11, h 14) at 227-227" and 230-230"; and N-glycosylation sites (H CH2 N84.4>A) at 298 and 298'.

[0273] In some embodiments, the anti-PD-Ll antibody is an anti-PD-Ll biosimilar monoclonal antibody approved by drug regulatory authorities with reference to atezolizumab. In some embodiments, the biosimilar comprises an anti-PD-Ll antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is atezolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-Ll antibody authorized or submitted for authorization, wherein the anti-PD-Ll antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is atezolizumab. The anti-PD-Ll antibody may be authorized by a drug regulatory authority such as the U.S. FDA and / or the European Union’s EMA. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is atezolizumab. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is atezolizumab.

[0274] In some embodiments, PD-L1 inhibitors include those antibodies described in U.S. Patent Application Publication No. US 2014 / 0341917 Al, the disclosure of which is incorporated by reference herein. In other embodiments, antibodies that compete with any of these antibodies for binding to PD-L1 are also included. In some embodiments, the anti-PD- Ll antibody is MDX-1105, also known as BMS-935559, which is disclosed in U.S. Patent No. US 7,943,743, the disclosures of which are incorporated by reference herein. In someAtorney Docket No. : 5133- WO embodiments, the anti-PD-Ll antibody is selected from the anti-PD-Ll antibodies disclosed in U.S. Patent No. US 7,943,743, which are incorporated by reference herein.

[0275] In some embodiments, the PD-L1 inhibitor is a commercially-available monoclonal antibody, such as INVIVOMAB anti-m-PD-Ll clone 10F.9G2 (Catalog # BE0101, Bio X Cell, Inc., West Lebanon, NH, USA). In some embodiments, the anti-PD-Ll antibody is a commercially-available monoclonal antibody, such as AFFYMETRIX EBIOSCIENCE (MIH1). A number of commercially-available anti-PD-Ll antibodies are known to one of ordinary skill in the art.

[0276] In some embodiments, the PD-L2 inhibitor is a commercially-available monoclonal antibody, such as BIOLEGEND 24F. IOC 12 Mouse IgG2a, K isotype (catalog # 329602 Biolegend, Inc., San Diego, CA), SIGMA anti-PD-L2 antibody (catalog # SAB3500395, Sigma-Aldrich Co., St. Louis, MO), or other commercially-available anti-PD-L2 antibodies known to one of ordinary skill in the art.2. Combinations with LAG-3 Inhibitors

[0277] In some embodiments, the TIL therapy provided to patients with cancer may include treatment with therapeutic populations of TILs alone or may include a combination treatment including TILs and one or more LAG-3 inhibitors. In some embodiments, the TIL therapy provided to patients with cancer may include treatment with therapeutic populations of TILs alone or may include a combination treatment including TILs and one or more LAG-3 inhibitors and one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0278] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0279] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating epithelial cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating epithelial cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.Atorney Docket No. : 5133- WO

[0280] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating methothelioma cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating methothelioma cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0281] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating endothelial cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating endothelial cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0282] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD- L1 inhibitors for treating cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating cancer in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T- lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof. In some embodiments, the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triplenegative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junctionAtorney Docket No. : 5133- WO cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC). hypopharynx cancer, larynx cancer, nasopharynx cancer, oropharynx cancer, and pharynx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T- lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0283] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0284] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating melanoma in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other w ords, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments,Atorney Docket No. : 5133- WO immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof

[0285] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating a patient or subject with unresectable or metastatic melanoma previously treated with a PD-1 blocking antibody, and if BRAF V600 mutation positive, a BRAF inhibitor with or without a MEK inhibitor. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating a patient or subject with unresectable or metastatic melanoma previously treated with a PD-1 blocking antibody, and if BRAF V600 mutation positive, a BRAF inhibitor with or without a MEK inhibitor, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0286] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD- 1 inhibitors for treating a patient or subject with unresectable or metastatic melanoma previously treated with a PD-1 blocking antibody, and if BRAF V600 mutation positive, a BRAF inhibitor with or without a MEK inhibitor. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating a patient or subject with unresectable or metastatic melanoma previously treated with a PD-1 blocking antibody, and if BRAF V600 mutation positive, a BRAF inhibitor with or without a MEK inhibitor. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating a patient or subject with unresectable or metastatic melanoma previously treated with a PD-1 blocking antibody, and if BRAF V600 mutation positive, a BRAF inhibitor with or without a MEK inhibitor. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyteAtorney Docket No. : 5133- WO associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0287] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating mucosal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating mucosal melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0288] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating mucosal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating mucosal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating mucosal melanoma in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0289] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating uveal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating uveal melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0290] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating uveal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors andAtorney Docket No. : 5133- WO one or more PD-L1 inhibitors for treating uveal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating uveal melanoma in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0291] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating choroidal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating choroidal melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0292] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating choroidal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating choroidal melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating choroidal melanoma in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0293] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating ciliary7body melanoma in aAtorney Docket No. : 5133- WO patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating ciliary body melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0294] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating ciliary body melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating ciliary body melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating ciliary body melanoma in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors: in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0295] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating iris melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating iris melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0296] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating iris melanoma in a patient or subj ect. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating iris melanoma in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating iris melanoma in a patient or subject. In some embodiments, the patientAtorney Docket No. : 5133- WO or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0297] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating NSCLC in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating NSCLC in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0298] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating NSCLC in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD- L1 inhibitors for treating NSCLC in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating NSCLC in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T- lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0299] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating HNSCC in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0300] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treatingAtorney Docket No. : 5133- WOHNSCC in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD- L1 inhibitors for treating HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating HNSCC in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T- lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0301] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating endometrial cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating endometrial cancer in a patient or subj ect, further combining with one or more PD- 1 inhibitors and / or one or more PD-L1 inhibitors.

[0302] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating endometrial cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating endometrial cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating endometrial cancer in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.Atorney Docket No. : 5133- WO

[0303] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors for treating cervical cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0304] In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for treating cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for treating cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein can be administered in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for treating cervical cancer in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0305] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of cancer in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0306] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors are for use in the treatment of cancer in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors are for use in the treatment of cancer in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors are for use in the treatment of cancerAtorney Docket No. : 5133- WO in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T- lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0307] In some embodiments, the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypophary nx cancer, lary nx cancer, nasopharynx cancer, oropharynx cancer, and pharynx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and smallcell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.Atorney Docket No. : 5133- WO

[0308] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of melanoma in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0309] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors are for use in the treatment of melanoma in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors are for use in the treatment of melanoma in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors are for use in the treatment of melanoma in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0310] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of HNSCC in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0311] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors are for use in the treatment of HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors are for use in the treatment of HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors are for use in the treatment ofAtorney Docket No. : 5133- WOHNSCC in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0312] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors are for use in the treatment of cervical cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0313] In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD- 1 inhibitors are for use in the treatment of cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors are for use in the treatment of cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors are for use in the treatment of cervical cancer in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0314] In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of cancer in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of cancer in a patient orAtorney Docket No. : 5133- WO subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0315] In some embodiments, TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD- 1 inhibitors for the treatment of cancer in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for the treatment of cancer in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD- 1 inhibitors and one or more PD-L 1 inhibitors for the treatment of cancer in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T- lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof. In some embodiments, the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triplenegative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypopharynx cancer, larynx cancer, nasopharynx cancer, orophary nx cancer, and phary nx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterineAtorney Docket No. : 5133- WO cancer, and vaginal cancer. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is immune checkpoint inhibitor naive patient or subject. In some embodiments, immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T- lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0316] In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of melanoma in a patient or subject. In some embodiments, t the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of melanoma in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0317] In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for the treatment of melanoma in a patient or subject. In some embodiments, the TILs produced as described herein in are used combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for the treatment of melanoma in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for the treatment of melanoma in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death- 1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0318] In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of HNSCC in a patient orAtorney Docket No. : 5133- WO subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0319] In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for the treatment of HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for the treatment of HNSCC in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for the treatment of HNSCC in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0320] In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors for the treatment of cervical cancer in a patient or subject, further combining with one or more PD-1 inhibitors and / or one or more PD-L1 inhibitors.

[0321] In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors for the treatment of cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-L1 inhibitors for the treatment of cervical cancer in a patient or subject. In some embodiments, the TILs produced as described herein are used in combination with one or more LAG-3 inhibitors and one or more PD-1 inhibitors and one or more PD-L1 inhibitors for the treatment of cervical cancer in a patient or subject. In some embodiments, the patient or subject is not previously treated with one or more immune checkpoint inhibitors; in other words, the patient or subject is an immune checkpoint inhibitor naive patient or subject. InAtorney Docket No. : 5133- WO some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a Programmed Cell Death-1 (PD-1) inhibitor, a Programmed Cell Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, and combinations thereof.

[0322] LAG-3 is an inhibitory immune checkpoint protein expressed on the surface of certain T-cells (such as CD4+ T cells, CD8+ T cells, regulatory T-cells (treg) and natural killer cells (NK)). When T-cells are activated, the expression of LAG-3 becomes more prominent. (Su et al., Front. Pharmacol. 2023; 14: 1349081). Lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells after major histocompatibility complex (MHC) class II ligation. Although its mechanism remains unclear, its modulation causes a negative regulatory effect over T cell function, preventing tissue damage and autoimmunity. LAG-3 and PD-1 are frequently co-expressed and upregulated on TILs. leading to immune exhaustion and tumor growth. Thus, LAG-3 blockade improves anti-tumor responses. See, e.g., Marin-Acevedo et al.. Journal of Hematology & Oncology 2018; 11 :39. A number of fully human anti-human LAG-3 monoclonal antibodies (mAbs) have been studied in clinical trials for the treatment of various types of solid tumors, including, but not limited to, relatlimab, favezelimab (MK-4280), fianlimab, Sym022, GSK2831781 (1MP731), INCAGN02385, TSR-033. and leramihmab (LAG525). Eftilagimod alpha (IMP321), a soluble LAG-3 protein as disclosed in US9579382B2 (incorporated herein by reference), is a major histocompatibility complex class II agonist activating antigen-presenting cells which leads to greater systemic type 1 T helper response and more cytotoxic CD8+ T-cell activation.

[0323] In some embodiments, a LAG-3 inhibitor may be any LAG-3 inhibitor or LAG-3 blocker known in the art. In particular, it is one of the LAG-3 inhibitors or blockers described in more detail in the following paragraphs. The terms “inhibitor,” “antagonist,” and “blocker” are used interchangeably herein in reference to LAG-3 inhibitors. For avoidance of doubt, references herein to a LAG-3 inhibitor that is an antibody may refer to a compound or antigen-binding fragments, variants, conjugates, or biosimilars thereof. For avoidance of doubt, references herein to a LAG-3 inhibitor may also refer to a LAG-3 fusion protein, a LAG-3 peptide, or a small molecule compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.Atorney Docket No. : 5133- WO

[0324] Suitable LAG-3 inhibitors for use in the methods of the disclosure include, without limitation, anti-LAG-3 antibodies, human anti-LAG-3 antibodies, mouse anti-LAG-3 antibodies, mammalian anti-LAG-3 antibodies, humanized anti-LAG-3 antibodies, monoclonal anti-LAG-3 antibodies, polyclonal anti-LAG-3 antibodies, chimeric anti-LAG-3 antibodies, anti-LAG-3 adnectins, anti-LAG-3 domain antibodies, single chain anti-LAG-3 fragments, heavy chain anti-LAG-3 fragments, light chain anti-LAG-3 fragments, LAG-3 fusion proteins, and LAG-3 peptides.

[0325] In some embodiments, the LAG-3 inhibitor is relatlimab, or biosimilars, antigenbinding fragments, conjugates, or variants thereof. As is known in the art, relatlimab refers to an anti-LAG-3 antibody, a fully human IgG4i< antibody derived from a transgenic mouse with human genes encoding heavy and light chains to generate a functional human repertoire. Thudium, et al., Cancer Immunol. Res., 2022, 10(10): 1175-1189, the content of which is herein incorporated by reference in its entirety. Relatlimab can also be referred to by its CAS Registry Number 1673516-98-7, and in PCT Publication Numbers WO 2014 / 008218 and WO 2015 / 042246, the content of which is incorporated herein by reference in their entireties. It is disclosed as antibody 25F7. A pharmaceutical composition of ipilimumab includes all pharmaceutically acceptable compositions containing ipilimumab and one or more diluents, vehicles, or excipients. Relatlimab can be administered intravenously (IV).

[0326] In some embodiments, the LAG-3 inhibitor is a LAG-3 biosimilar monoclonal antibody approved by drug regulatory authorities with reference to ipilimumab. In some embodiments, the biosimilar comprises an anti-LAG-3 antibody comprising an amino acid sequence which has at least 97% sequence identity', e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-LAG-3 antibody authorized or submitted for authorization, wherein the anti-LAG-3 antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is ipilimumab. The anti-LAG-3 antibody may be authorized by a drug regulatory authority suchAtorney Docket No. : 5133- WO as the U.S. FDA and / or the European Union’s EMA. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is relatlimab. In some embodiments, the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is relatlimab.

[0327] In some embodiments, the LAG-3 inhibitor is relatlimab or a biosimilar thereof, and relatlimab is administered at a dose of about 0.5 mg / kg to about 10 mg / kg. In some embodiments, the LAG-3 inhibitor is relatlimab or a biosimilar thereof, and relatlimab is administered at a dose of about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, or about 10 mg / kg. In some embodiments, the relatlimab administration is begun 1, 2. 3, 4, or 5 weeks pre-resection (z.e., prior to obtaining the tumor sample from the subject or patient). In some embodiments, the relatlimab administration is begun 1, 2, or 3 weeks pre-resection (z.e., prior to obtaining the tumor sample from the subject or patient). In some embodiments, relatlimab is administered contemporaneously with the population of TILs. In some embodiments, relatlimab is administered not contemporaneously with the population of TILs. In some embodiments, relatlimab is administered after administering the population of TILs. In some embodiments, relatlimab is administered at least one week, at least two weeks, at least three weeks, at least four weeks, after administering the population of TILs. In some embodiments, relatlimab is maintained after administering the population of TILs.

[0328] In some embodiments, the LAG-3 inhibitor is relatlimab or a biosimilar thereof, and relatlimab is administered at a dose of about 10 mg to about 300 mg. In some embodiments, the LAG-3 inhibitor is relatlimab or a biosimilar thereof, and relatlimab is administered at a dose of about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg. about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg. In some embodiments, theAtorney Docket No. : 5133- WO relatlimab administration is begun 1, 2, 3, 4, or 5 weeks pre-resection (i.e., prior to obtaining the tumor sample from the subject or patient). In some embodiments, the relatlimab administration is begun 1, 2. or 3 weeks pre-resection (z.e., prior to obtaining the tumor sample from the subject or patient). In some embodiments, relatlimab is administered contemporaneously with the population of TILs. In some embodiments, relatlimab is administered not contemporaneously with the population of TILs. In some embodiments, relatlimab is administered after administering the population of TILs. In some embodiments, relatlimab is administered at least one week, at least two weeks, at least three weeks, at least four weeks, after administering the population of TILs. In some embodiments, relatlimab is maintained after administering the population of TILs.

[0329] In some embodiments, the LAG-3 inhibitor is relatlimab or a biosimilar thereof, and relatlimab is administered every week, even' 2 weeks, every' 3 weeks, every' 4 weeks, every' 5 weeks, or every 6 weeks. In some embodiments, the relatlimab administration is begun 1, 2, 3, 4, or 5 weeks pre-resection (z.e., prior to obtaining the tumor sample from the subject or patient). In some embodiments, the relatlimab administration is begun 1, 2, or 3 weeks pre- resection (z.e., prior to obtaining the tumor sample from the subject or patient). In some embodiments, relatlimab is administered contemporaneously with the population of TILs. In some embodiments, relatlimab is administered not contemporaneously with the population of TILs. In some embodiments, relatlimab is administered after administering the population of TILs. In some embodiments, relatlimab is administered at least one yveek, at least tyvo veeks, at least three weeks, at least four weeks, after administering the population of TILs. In some embodiments, relatlimab is maintained after administering the population of TILs.

[0330] In some embodiments, a combination therapy of relatlimab and nivolumab (OPDUALAG™) is administered to treat unresectable or metastatic melanoma. In some embodiments, relatlimab is administered to treat unresectable or metastatic melanoma at about 160 mg immediately follo ving nivolumab 480 mg on the same day, every' yveek, every' 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every' 6 weeks for 4 doses. In some embodiments, after completing 4 doses of the combination, nivolumab can be administered as a single agent according to standard dosing regimens for unresectable or metastatic melanoma. In some embodiments, the relatlimab and nivolumab administration is begun 1, 2, 3, 4, or 5 weeks pre-resection (i.e.. prior to obtaining the tumor sample from the subject or patient). In some embodiments, the relatlimab and nivolumab administration is begun 1, 2. orAtorney Docket No. : 5133- WO3 weeks pre-resection ( / . e. , prior to obtaining the tumor sample from the subject or patient). In some embodiments, relatlimab and nivolumab are administered contemporaneously with the ...

Claims

1. Atorney Docket No. : 5133- WOWHAT IS CLAIMED IS:

1. A method of treating a cancer in a patient in need thereof, comprising: i) administering a therapeutic population of tumor infiltrating lymphocytes (TILs) to the patient; and ii) administering a LAG-3 inhibitor to the patient.

2. The method of claim 1, wherein the LAG-3 inhibitor is an anti-LAG-3 antibody.

3. The method of claim 2, wherein the anti-LAG-3 antibody is selected from the group consisting of relatlimab, favezelimab (MK.-4280), fianlimab, Sym022, GSK2831781 (IMP731), INCAGN02385, TSR-033, ieramilimab (LAG525), and a biosimilar thereof.

4. The method of claim 3, wherein the anti-LAG-3 antibody is relatlimab or a biosimilar thereof.

5. The method of claim 3, wherein the anti-LAG-3 antibody is favezelimab (MK-4280) or a biosimilar thereof.

6. The method of claim 3, wherein the anti-LAG-3 antibody is fianlimab or a biosimilar thereof.

7. The method of any one of claims 1-6, further comprising administering a PD-1 inhibitor and / or a PD-L1 inhibitor to the patient.

8. The method of claim 7, wherein the PD-1 inhibitor is an anti-PD-1 antibody and the PD- L1 inhibitor is an anti-PD-Ll antibody.

9. The method of claim 8, wherein the anti-PD-1 antibody or the anti-PD-Ll antibody is selected from the group consisting of nivolumab (Opdivo), pembrolizumab (Keytruda), cemiplimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), and a biosimilar thereof.

10. The method of claim 9, wherein the anti-PD-1 antibody is nivolumab or a biosimilar thereof.

11. The method of claim 9, wherein the anti-PD-1 antibody is pembrolizumab or a biosimilar thereof.Atorney Docket No. : 5133- WO12. The method of claim 9, wherein the anti-PD-1 antibody is cemiplimab or a biosimilar thereof.

13. The method of claim 10, wherein the anti-LAG-3 antibody is relatlimab or a biosimilar thereof and the anti-PD-1 antibody is nivolumab or a biosimilar thereof.

14. The method of claim 13, wherein relatlimab or a biosimilar thereof is administered at a dosage of about 160 mg and nivolumab or a biosimilar thereof is administered at a dosage of about 480 mg, infused over 30 minutes.

15. The method of claim 13 or 14. wherein relatlimab or a biosimilar thereof and nivolumab or a biosimilar thereof are administered weekly, once every two weeks, or once every three weeks.

16. The method of any one of claims 7-15, wherein the LAG-3 inhibitor and / or the PD-l / PD- L1 inhibitor is administered contemporaneously with the population of TILs.

17. The method of any one of claims 7-15, wherein the LAG-3 inhibitor and / or the PD-l / PD- L1 inhibitor is not administered contemporaneously with the population of TILs.

18. The method of any one of claims 7-15, wherein the LAG-3 inhibitor and / or the PD-l / PD- L1 inhibitor is administered after administering the population of TILs.

19. The method of any one of claims 7-15, wherein the LAG-3 inhibitor and / or the PD-l / PD- L1 inhibitor is administered at least one week after administering the population of TILs.

20. The method of any one of claims 7-19, wherein the LAG-3 inhibitor and / or the PD-l / PD- L1 inhibitor is maintained after administering the population of TILs.

21. The method of any one of claims 1-20, wherein the patient is refractory to pre-treatment with a LAG-3 inhibitor or a biosimilar thereof.

22. The method of any one of claims 1-21, wherein the patient is refractory to pre-treatment with a PD-1 inhibitor and / or a PD-L1 inhibitor or a biosimilar thereof.

23. The method of claim 22, wherein the patient has been previously treated with a PD-1 inhibitor or a biosimilar thereof.

24. The method of claim 23, wherein the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and biosimilars thereof.

25. The method of any one of claims 22-24, wherein the patient has been previously treatedAtorney Docket No. : 5133- WO with a PD-L1 inhibitor or a biosimilar thereof.

26. The method of claim 25, wherein the PD-L1 inhibitor is selected from the group consisting of avelumab, atezolizumab, durvalumab, and biosimilars thereof.

27. The method of any one of claims 1-20, wherein the patient is naive to an immune checkpoint inhibitor (ICI) treatment.

28. The method of claim 27, wherein the ICI treatment comprises a PD-1 inhibitor and / or a PD-L1 inhibitor.

29. The method of claim 28, wherein the PD-1 inhibitor and / or PD-L1 inhibitor is selected from the group consisting of nivolumab. pembrolizumab, cemiplimab, avelumab, atezolizumab, durvalumab, and biosimilars thereof.

30. The method of any one of claims 27-29. wherein the ICI treatment comprises a LAG-3 inhibitor.

31. The method of claim 30, wherein the LAG-3 inhibitor is selected from the group consisting of relatlimab, favezelimab (MK-4280), fianlimab, Sym022, GSK2831781 (IMP731), INCAGN02385, TSR-033, ieramilimab (LAG525), and a biosimilar thereof.

32. The method of any one of claims 27-31, wherein the ICI treatment comprises a CTLA-4 inhibitor.

33. The method of claim 32, wherein the CTLA-4 inhibitor is selected from the group consisting of ipilimumab, tremelimumab, zalifrelimab, AGEN1181, BMS-986218, BCD- 145, ONC-392, CSI002. REGN4659, ADG116. and a biosimilar thereof.

34. The method of any one of claims 1-33, wherein the patient has been previously treated with a chemotherapeutic regimen.

35. The method of claim 34. wherein the chemotherapeutic regimen comprises carboplatin, paclitaxel, pemetrexed, and / or cisplatin.

36. The method of any one of claims 1-33, wherein the patient has not been previously treated with a chemotherapeutic regimen.

37. The method of any one of claims 1-36, wherein the patient has been previously treated with an angiogenesis inhibitor.

38. The method of claim 37, wherein the angiogenesis inhibitor is bevacizumab.Atorney Docket No. : 5133- WO39. The method of any one of claims 1-36, wherein the patient has not been previously treated with an angiogenesis inhibitor.

40. The method of any one of claims 1-39, wherein the therapeutic population of TILs is produced by:(a) obtaining and / or receiving a first population of TILs from a tumor resected from the patient;(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs. wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs; and(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs comprises the therapeutic population of TILs.

41. The method of claim 40. further comprising:(d) harvesting therapeutic population of TILs.

42. The method of claim 41, further comprising:(e) transferring the harvested therapeutic population of TILs into an infusion bag.

43. The method of claim 42, further comprising:(f) cryopreserving the infusion bag comprising the harvested therapeutic population of TILs using a cryopreservation process.

44. The method of any one of claims 40-43, wherein the first expansion is performed over a period of about 3-11 days.

45. The method of any one of claims 40-43, wherein the first expansion is performed over a period of about 11 days.

46. The method of any one of claims 40-45, wherein the second expansion is performed over a period of about 7-11 days.

47. The method of any one of claims 40-45, wherein the second expansion is performed over a period of about 11 days.Atorney Docket No. : 5133- WO48. The method of any one of claims 40-43, wherein the first expansion is performed over a period of about 11 days, and the second expansion is performed over a period of about 11 days.

49. The method of any one of claims 40-48, wherein step (b) and step (c) are performed in a closed system, wherein the transition from step (b) to step (c) occurs without opening the closed system.

50. The method of claim 49, wherein the transition from step (c) to step (d) occurs without opening the closed system.

51. The method of claim 49 or 50, wherein the transition from step (d) to step (e) occurs without opening the closed system.

52. The method of any one of claims 1-51, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

53. The method of claim 52, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for five days.

54. The method of claim 52, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

55. The method of any one of claims 53 or 54, wherein the cyclophosphamide is administered with mesna.

56. The method of any one of claims 1-55, further comprising the step of treating the patient with an IL-2 regimen starting on the day after the administration of the TILs to the patient.

57. The method of any one of claims 1-55, further comprising the step of treating the patient with an IL-2 regimen starting on the same day as administration of the TILs to the patient.

58. The method of claim 56 or 57, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 lU / kg of aldesleukin, or a biosimilar or variant thereof,Atorney Docket No. : 5133- WO administered as a 15 -minute bolus intravenous infusion every' eight hours until tolerance.

59. The method according to any one of claims 1-58, wherein a therapeutically effective population of TILs is administered and comprises from about 1 xlO9to about lOxlO10TILs.

60. The method according to any one of claims 1-58, wherein a therapeutically effective population of TILs is administered and comprises from about 7.5 x lO9to about 7.5 x lO10TILs.

61. The method of any one of claims 1-60, wherein the cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system associated cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma. and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypopharynx cancer, larynx cancer, nasopharynx cancer, oropharynx cancer, and pharynx cancer), kidney cancer, liver cancer, lung cancer (including non-small-cell lung cancer (NSCLC) and small-cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, choroidal melanoma, ciliary body melanoma, or ins melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, renal cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer.

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