Expansion processes for til product enriched with neoantigen- reactive t cells (NARTS)

Abstract

Provided herein are methods for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: generating a plurality of TIL populations by fractioning a tumor sample; selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); and preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate the TIL product enriched with NARTs. Further provided is a method for assessing tumor reactivity of a population of T cells, comprising measurement of the expression level of a panel of target genes of the population of T cells.

Description

EXPANSION PROCESSES FOR TH PRODUCT ENRICHED WITH NEOANTIGEN- RREACTIVE T CELLS (NARTS)BACKGROUND

[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. 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. 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. Combination studies with single immune checkpoint inhibitors have also been described, but further studies are ongoing and additional methods of treatment are needed.

[0002] The current TIL expansion process results in both tumor specific TILs (TS-TILs) and bystander TILs in the expanded TIL product. Studies show that frequency of TS-TILs in the expanded TIL product predicts responsiveness in melanoma patients receiving TIL therapy. Bystander T cells can have a Treg-like effect of stealing IL-2 and homeostatic cytokines from effector TS-TILs. Bystander TILs also outcompete TS-TIL for nutrients and oxygen during the REP period, reducing their total numbers because: a) bystander TILs tend to be younger and fitter (less-differentiated) T cells and thus have more proliferative potential; and b) TIL growth is dependent on metabolic state and phenotypic state of TILs, i.e. expression of costimulatory receptors. Bystander TILs take up valuable tumor real-estate after adoptive transfer and create a "traffic jam" getting to tumor.

[0003] The present disclosure provides an improved expansion process for producing a TIL product enriched with neoantigen-reactive T cells (NARTs), and reduced bystander T cells.BRIEF SUMMARY

[0004] In some embodiments, a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs) is provided.

[0005] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a. Generating a plurality of TIL populations by fractioning a tumor sample; b. Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); and c. Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate the TIL product enriched with NARTs.

[0006] In some embodiments, profiling the plurality of TIL populations comprises quantification of T cell DNA copy number of the plurality of TIL populations. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells. In some embodiments, the unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements is TRAB. In embodiments, the regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells is TRBC. In embodiments, the reference DNA marker existing as a diploid in all cells is RPP30 (Ribonuclease P protein subunit p30).

[0007] In some embodiments, profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations. In some embodiments, the panel of target genes comprises one or more genes selected from CXCL13 (C-X-C motif chemokine ligand 13), CD200 (Cluster of Differentiation 200), TRBC (T Cell Receptor Beta Constant; e.g., TRBC1 and / or TRBC2), YWHAZ (tyrosine 3- monooxygenase / tryptophan 5-monooxygenase activation protein zeta), CD20 (Cluster of Differentiation 20), and CD19 (Cluster of Differentiation 19). In some embodiments, measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes. In some embodiments, the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI). In some embodiments, the ICI comprises a CD38 inhibitor, a CTLA-4 (Cytotoxic T-lymphocyte associated protein 4) inhibitor and / or aPD-1 (Programmed Cell Death Protein 1) inhibitor and / or PD-L1 (Programmed cell death ligand 1) inhibitor. In some embodiments, the first cell culture medium in step (c) further comprises soluble IL-12 (interleukin 12) and / or a 4-1BB (4-immunoglobulin superfamily- 1B) agonist. In some embodiments, the 4-1BB agonist is urelumab.

[0008] In some embodiments, the method further comprises selecting CD137+, CD200+ and / or CD103+ TILs from the TIL product enriched with NARTs in step (c) to generate a positively-selected TIL product. In some embodiments, the method further comprises removing PD-1H|TILs from the TIL product enriched with NARTs in step (c) or the positively- selected TIL product to generate a bystander-depleted TIL product.

[0009] In some embodiments, the method further comprises: d) culturing the TIL product in a second cell culture medium in the presence of artificial antigen presenting cells (aAPCs). In some embodiments, the aAPCs are acellular-based or cellular-based. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises L-arginine. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ (nicotinamide adenine dinucleotide) booster. In some embodiments, the NAD+ booster is one or more of L-Trp, NR (nicotinamide riboside), NMN (nicotinamide mononucleotide), NAD+, NAM (nicotinamide) or P7C3 activator. In some embodiments, the second cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L-lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate ((3- OHB), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate. In some embodiments, the second cell culture medium comprises a mitophagy activator. In some embodiments, the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

[0010] In some embodiments, step (a) comprises fractioning the tumor sample into multiple tumor fragments. In some embodiments, the tumor sample is fractioned into 2 to 100 tumor fragments. In some embodiments, step (a) comprises digesting the tumor sample into a tumor digest.

[0011] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising:a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) selecting CD137+, CD200+ and / or CD103+ TILs from the first population of TILs to generate a second population of TILs; and e) Culturing the second population of TILs in a second cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

[0012] In some embodiments, profiling the plurality of TIL populations comprises quantification of T cell DNA copy number of the plurality of TIL populations. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells. In some embodiments, the unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements is TRAB, the regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells is TRBC, and the reference DNA marker existing as a diploid in all cells is RPP30. In some embodiments, profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations. In some embodiments, the panel of target genes comprises one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19. In some embodiments, measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes. In some embodiments, the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI). In some embodiments, the ICI comprises a CD38 inhibitor, a CTLA-4 inhibitor and / or a PD-1 / PD-L1 inhibitor. In some embodiments, the first cell culture medium in step (c) further comprises soluble IL-12 and / or a 4-1BB agonist. In some embodiments, the 4-1BB agonist is urelumab.

[0013] In some embodiments, the method further comprises removing PD-1HiTILs from the first or second population of TILs to generate a bystander-depleted TIL product.

[0014] In some embodiments, the aAPCs are acellular-based or cellular-based. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises L-arginine. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is one or more of L-Trp, NR, NMN, NAD+, NAM or P7C3 activator. In some embodiments, the second cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L- lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate (|3-OHB), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate. In some embodiments, the second cell culture medium comprises a mitophagy activator. In some embodiments, the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

[0015] In some embodiments, step (a) comprises fractioning the tumor sample into multiple tumor fragments. In some embodiments, the tumor sample is fractioned into 2 to 100 tumor fragments. In some embodiments, step (a) comprises digesting the tumor sample into a tumor digest.

[0016] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) Culturing the first population of TILs in a second cell culture medium to generate a second population of TILs; and e) Culturing the second population of TILs in a third cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

[0017] In some embodiments, profiling the plurality of TIL populations comprises quantification of T cell DNA copy number of the plurality of TIL populations. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells. In some embodiments, the unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements is TRAB, the regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells is TRBC, and the reference DNA marker existing as a diploid in all cells is RPP30. In some embodiments, profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations. In some embodiments, the panel of target genes comprises one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19. In some embodiments, measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes. In some embodiments, the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI). In some embodiments, the ICI comprises a CD38 inhibitor, a CTLA-4 inhibitor and / or a PD-1 / PD-L1 inhibitor. In some embodiments, the first cell culture medium in step (c) further comprises soluble IL-12 and / or a 4-1BB agonist. In some embodiments, the 4-1BB agonist is urelumab.

[0018] In some embodiments, the method further comprises selecting CD137+, CD200+ and / or CD103+ TILs from the first or second population of TILs to generate a positively- selected TIL product. In some embodiments, the method further comprises removing PD-1H|TILs from the first or second population of TILs or the positively-selected TIL product to generate a bystander-depleted TIL product.

[0019] In some embodiments, the aAPCs are acellular-based or cellular-based. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises L-arginine. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is one or more of L-Trp, NR, NMN, NAD+, NAM or P7C3 activator. In some embodiments, the second cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L-lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate (P-OHB), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate. In some embodiments, the second cell culture medium comprises a mitophagy activator. In some embodiments, the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

[0020] In some embodiments, step (a) comprises fractioning the tumor sample into multiple tumor fragments. In some embodiments, the tumor sample is fractioned into 2-100 tumor fragments. In some embodiments, step (a) comprises digesting the tumor sample into a tumor digest.

[0021] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) Culturing the first population of TILs in a second cell culture medium to generate a second population of TILs; e) selecting CD137+, CD200+ and / or CD103+ TILs from the second population of TILs to generate a third population of TILs; and f) Culturing the third population of TILs in a third cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

[0022] In some embodiments, profiling the plurality of TIL populations comprises quantification of T cell DNA copy number of the plurality of TIL populations. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells. In some embodiments, the unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJrearrangements is TRAB, the regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells is TRBC, and the reference DNA marker existing as a diploid in all cells is RPP30. In some embodiments, profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations. In some embodiments, the panel of target genes comprises one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19. In some embodiments, measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes. In some embodiments, the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI). In some embodiments, the ICI comprises a CD38 inhibitor, a CTLA-4 inhibitor and / or a PD-1 / PD-L1 inhibitor. In some embodiments, the first cell culture medium in step (c) further comprises soluble IL-12 and / or a 4-1BB agonist. In some embodiments, the 4-1BB agonist is urelumab.

[0023] In some embodiments, the method further comprises removing PD-lHiTILs from the first, second or third population of TILs to generate a bystander-depleted TIL product.

[0024] In some embodiments, the aAPCs are acellular-based or cellular-based. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises L-arginine. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the second cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L- lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate (3-OHB), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate. In some embodiments, the second cell culture medium comprises a mitophagy activator. In some embodiments, the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

[0025] In some embodiments, step (a) comprises fractioning the tumor sample into multiple tumor fragments. In some embodiments, the tumor sample is fractioned into 2-100 tumor fragments. In some embodiments, step (a) comprises digesting the tumor sample into a tumor digest.

[0026] In some embodiments, provided herein is a method for assessing tumor reactivity of a population of T cells, comprising measurement of the expression level of a panel of target genes of the population of T cells. In some embodiments, the panel of target genes comprises one or more genes selected from CXCL13, CD200, TRBC, YWHAZ, CD20, and CD19.

[0027] In some embodiments, measurement of the expression level of the panel of target genes comprises quantification of the mRNA molecules of each of the target genes. In some embodiments, quantification of the mRNA molecules of each of the target genes comprises determining the copy number of the mRNA molecules of each of the target genes. In some embodiments, determining the copy number of the mRNA molecules comprises reverse transcription of the mRNA molecules into cDNA molecules. In some embodiments, the cDNA molecules are quantified by digital PCR (dPCR). In some embodiments, the method further comprises quantification of T cell DNA copy number of the population of T cells. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells. In some embodiments, the unique T cell DNA marker that is bial lelical ly absent in T cells due to VDJ rearrangements is TRAB. In embodiments, the regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells is TRBC, and the reference DNA marker existing as a diploid in all cells is RPP30.

[0028] In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs). In some embodiments, the population of T cells is admixed with a population of non-T cells. In some embodiments, the non-T cells are tumor cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1: Outlines the "TS-TIL" (4-1BB selection) process for producing tumor reactive TILs.

[0030] Figure 2: Shows tumor reactivity was enhanced when TILs were cultured with ITIL cytokines in the presence of interferon gamma (I FNy).

[0031] Figure 3A-3B: Shows 4-1BB expression was found to be maximal 24 hours after plating the tumor digest (A), and increase with the provision of IFNy to the cell culture media (TS-TIL condition) (B).

[0032] Figure 4: Outlines several processes that were analyzed for their capacity to enrich tumor-reactive CD8 TILs.

[0033] Figure 5 : Shows ~10x improvement in product potency of TILs made by the CD137 sorted process.

[0034] Figure 6: Shows enrichment for tumor reactive TILs made by the TS-TIL (4-1BB sort / lx REP) process.

[0035] Figure 7 : Shows both CD137 (4-1BB) sorting conditions provided the best enrichment for tumor-reactive CD8s (IFNy +CD107a+) TILs.

[0036] Figure 8: Shows 60-fold improvement for tumor-reactive CD8s (IFNy +CD107a+) TILs by CD137 (4-1BB) sorting condition.

[0037] Figure 9 : Shows a significant increase in percentage of specific tumor reactive TILs in the TS-TIL product (~7%) vs the CTRL product (~1%).

[0038] Figure 10: Shows the total number of CDS tumor-reactive TILs generated from the TS-TIL process is, on average 7-fold, greater than the total number of CD8 tumor-reactive TILs generated from the CTRL process.

[0039] Figure 11: Shows the results for total viable cells and fold expansion as a function of REP conditions.

[0040] Figure 12: Shows the total yield of CD8+ tumor-reactive TIL (as assessed by autologous digest coculture ICS) as a function of sort and REP conditions.

[0041] Figure 13: Shows that the majority of cells was found to be Tern "effector memory" cells, with minimal differences among the TILs made from different processes.

[0042] Figure 14: Shows that TelL-15 activated T cells in cis manner only.

[0043] Figure 15: Panels A-D show that L-Arginine enhances expansion and cell recovery post thaw during Invigo-T REP.

[0044] Figure 16: Panels A-C show that L-Arginine provides better phenotype and killing capacity.

[0045] Figure 17: Panels A-C show that NAD+ synergies with L-Arginine in boosting metabolism and providing a unique T cell signature.

[0046] Figure 18: Panels A and B show that a wide range of T cell fractions detected (10- 40%) in a lung tumor (A) and a narrow range of T cell fractions was detected (5-20%) in a pancreatic tumor (B) using dPCR.

[0047] Figure 19: Shows results of TIL quantification by dPCR performed in melanoma FFPE blocks from patients that were responders or non-responders.

[0048] Figure 20: Shows T cell fraction (upper panel) and tumor reactivity (lower panel) profiles across fragments.DETAILED DESCRIPTIONI. Introduction

[0049] Provided herein are methods of producing TILs via (i) pre-REP stimulation with a combination of interferon gamma (IFN-y) and anti-PD-1, with or without CD40 agonism, and with or without CTLA-4 agonism (ii) adding various cytokine combinations such as IL-15, IL- 21, low-concentration IL-2, with or without AKT inhibition (AKTi), during REP expansion and / or pre-REP expansion, and / or (iii) low-concentration IL-2 and AKTi during REP expansion and / or pre-REP expansion. Also provided herein are methods of treatment using such TILs.Definitions

[0050] 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.

[0051] 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 some embodiments, for example, a plurality of TILs) to a subject so that both activepharmaceutical 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.

[0052] The term "in vivo’’ refers to an event that takes place in a subject's body.

[0053] The term "in 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.

[0054] 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.

[0055] The term "rapid expansion" means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein.

[0056] 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. "Primary TILs" 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 and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations can include genetically modified TILs.

[0057] 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 ( IFN y) 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.

[0058] The term "CD137" refers to a TNFR-family member with costimulatory function. CD137 is also called 4-1BB or TNFSFR9. It was originally identified as an inducible molecule expressed on activated mouse and human CD8+ and CD4+ T-cells (Watts, 2005, Annu Rev Immunol, 23: 23-68; Vinay et al.et al., 1998, Semin Immunol, 10: 481-9; Kwon and Weissman, 1989, Proc Natl Acad Sci USA, 86: 1963-7). CD137 signaling regulates T-cell proliferation and survival, particularly within the T-cell memory pool, can upregulate Bcl- XL anti-apoptotic protein expression, and supports CD8+ T-cell expansion.

[0059] By "CD39 / CD69 double negative and / or CD39LO / CD69LO TILs" or "CD39 / CD69 double negative and / or CD39LO / CD69LO TIL population" or grammatical variants of either of the foregoing is meant TILs or a population of TILs that display undetectable, lower, or reduced levels of the cell surface proteins CD39 and CD69 on average compared to any TILs / population of TILs from which the referenced TILs or population of TILs is obtained.

[0060] By "CD39 / CD69 double negative and / or CD39LO / CD69LO enriched TILs" or "CD39 / CD69 double negative and / or CD39LO / CD69LO enriched TIL population" or grammatical variants of either of the foregoing is meant TILs or a population of TILs that has been enriched for TILs with undetectable, low, or reduced levels of the cell surface proteins CD39 and CD69 on average compared to any TILs / population of TILs from which thereferenced TILs or population of TILs is obtained. Any means of enrichment can be used to obtain CD39 / CD69 double negative and / or CD39LO / CD69LO enriched TILs, including sorting or selecting for CD39 / CD69 double negative and / or CD39LO / CD69LO TILs.

[0061] 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 106 to 1 X 1011 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 108 cells. REP expansion is generally done to provide populations of 1.5 x 109 to 1.5 x 1010 cells for infusion.

[0062] 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 cryopreservation 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.

[0063] 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.

[0064] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreservation 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 CS10 medium is a serum-free, animal component-free medium which comprises DMSO.

[0065] The term "central memory T cell" refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCRtriggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.

[0066] The term "effector memory T cell" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-y, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.

[0067] 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 disclosed herein. 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 no opened to the outside environment until the TILs are ready to be administered to the patient.

[0068] 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.

[0069] The terms "peripheral blood mononuclear cells" and "PBMCs" refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as an antigen presenting cell (PBMCs are a type of antigen- presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0070] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are separated from whole blood or apheresis product from a donor. In some embodiments, PBLs are separated from whole blood orapheresis product from a donor by positive or negative selection of a T cell phenotype, such as the T cell phenotype of CD3+ CD45+.

[0071] 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.

[0072] The term "OKT-3" (also referred to herein as "OKT3") 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 (ECACC) and assigned Catalogue No. 86022706.

[0073] 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 as 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, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The term IL-2also 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 N6 substituted with [(2,7- bis{[methylpoly(oxyethylene)]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 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 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 are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated by reference herein.

[0074] In some embodiments, an IL-2 form is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 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.

[0075] In some embodiments, the IL-2 form is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form is pegylated as disclosed in W02021050554, U.S. Patent Application Publication No. US 2020 / 0181220 Al or U.S. Patent Application Publication No. US 2020 / 0330601 Al, each of which is incorporated by reference herein in its entirety.

[0076] In some embodiments, an IL-2 form is nemvaleukin alfa, also known as ALKS-4230, which is available from Alkermes, Inc. 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 are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated by reference herein.

[0077] In some embodiments, an IL-2 form includes a 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 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 regulatory 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.

[0078] 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.

[0079] In some embodiments, the antibody cytokine engrafted protein comprises lgG.IL2F71A.Hl or lgG.IL2R67A.Hl of U.S. Patent Application Publication No. 2020 / 0270334 Al, or variants, derivatives, or fragments thereof, or conservative amino acid substitutions thereof, or proteins with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. 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 that a wild-type IL-2 molecule such as, but not limited to, aldesleukin or a comparable molecule.

[0080] 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 andBorish, 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 IIMHC expression, and induces class switching to IgE and IgGl expression from B cells. Recombinant human IL-4 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).

[0081] 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. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and 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 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).

[0082] The term "IL-15" (also referred to herein as "I L15") refers to the T cell growth factor known 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).

[0083] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human andmammalian 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 a 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).

[0084] When "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or "therapeutic amount" is indicated, the precise amount of the composition(s) 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 104 to 1011 cells / kg body weight (e.g., 105 to 106, 105 to 1010, 105 to 1011, 106 to 1010, 106 to 1011,107 to 1011, 107 to 1010, 108 to 1011, 108 to 1010, 109 to 1011, or 109 to 1010 cells / 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 tumorTILs (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.

[0085] The term "hematological malignancy", "hematologic malignancy" or terms of correlative meaning refer to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblasticleukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term "B cell hematological malignancy" refers to hematological malignancies that affect B cells.

[0086] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including liquid tumors circulating in peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0087] 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 cues that promote neoplastic transformation, support tumor growth 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.

[0088] In some embodiments, provided herein is a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with 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 U 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 intravenously at 720,000 lU / kg every 8 hours to physiologic tolerance.

[0089] 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.

[0090] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or combination of compounds 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 (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0091] 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 delivery of 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.

[0092] 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, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new 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).

[0093] 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 considering any conservative amino acid substitutions as part of the sequence identity. 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 identity include 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.

[0094] As used herein, the term "variant" encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody 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 antibody. The variant may comprise one or more conservative substitutions in its amino acidsequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0095] 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. "Primary TILs" 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 for example second expansion TILs or second additional expansion TILs.

[0096] 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, CDS, 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.

[0097] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between deoxyribonucleotide in the oligonucleotide.

[0098] The term "RNA" defines a molecule comprising at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide with a hydroxyl group at the 2' position of a b-D-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Nucleotides of the RNA molecules described herein may also comprise non-standard nucleotides, such as non-natu rally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0099] 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 is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0100] 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%, 5%, or 1% of a given value or range. The allowable 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 be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. 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 isnoted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[0101] 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 ordinarily 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 can, in alternate embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0102] 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 two 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 hypervariability, 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, FRS, CDRS, FR4. The 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 theimmunoglobulin 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.

[0103] 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 histocompatibility complex (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.

[0104] 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.

[0105] 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 morefragments of 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 "antigenbinding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, 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, 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). 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.

[0106] The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework 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-specific mutagenesis in vitro or by somatic mutation in vivo). The term "human antibody", as usedherein, 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.

[0107] 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.

[0108] 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.

[0109] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

[0110] 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."

[0111] 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.

[0112] 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), typically that of a human immunoglobulin. 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 Al, WO 1999 / 51642 Al, WO 99 / 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.

[0113] 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.

[0114] A "diabody" is a small antibody fragment with two antigen-binding sites. The fragments comprises a heavy chain variable domain (VH) 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. Sci. USA 1993, 90, 6444-6448.

[0115] 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 therebyeliminate 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 having increased bisecting GIcNac 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 fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 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 GIcNac 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.

[0116] "Pegylation" 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 or antibody fragment. Pegylation 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 (Cl-ClO)alkoxy- or aryloxy-polyethylene 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.

[0117] 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, asamended 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 medicinal 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 quality characteristics 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 safety profile 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 identity of 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-translationalmodifications, 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 not 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. Method of Assessing Tumor-Reactivity of a Population of T cells

[0118] Some embodiments disclosed herein provide a method for assessing tumor reactivity of a population of T cells, comprising measurement of the expression level of a panel of target genes of the population of T cells.

[0119] Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates where adaptive anti-tumour cellular and humoral responses can be elaborated. Initially described in non-small cell lung cancer, TLS have also been found in melanomas and sarcomas, and associated with improved response to immunotherapy. TLS are composed predominantly of aggregates of B cells and T cells, follicular dendritic cells (FDCs). The manipulation of TLS as a therapeutic strategy is now coming of age owing to the likely role of TLS in the improved survival of patients. See Teillaud, J L., et al., 2024, Nat Rev Cancer 24,629-646; Trub M, 2021, Front. Immunol. 12:674565; the contents of which are herein incorporated by reference in their entireties.

[0120] Therefore, to assess the tumor reactivity of TILs, a genetic panel of TLS markers that are overexpressed in tumor reactive TILs are designed using transcriptomic data. In some embodiments, the genetic panel comprises one or more genes selected from the list including CXCL13, CD200, TRBC, YWHAZ, CD19, CD20, etc. In some embodiments, the genetic panel comprises CXCL13. In some embodiments, the genetic panel comprises CD200. In some embodiments, the genetic panel comprises TRBC. In some embodiments, the genetic panel comprises YWHAZ. In some embodiments, the genetic panel comprises CD19. In some embodiments, the genetic panel comprises CD20. The expression level from the gene(s) may be measured by any known means. In embodiments, protein levels expressed from the gene(s) are measured. In embodiments, mRNA levels expressed from the gene(s) are measured.

[0121] In some embodiments, measurement of the expression level of the panel of target genes comprises quantification of the mRNA molecules of each of the target genes. In some embodiments, quantification of the mRNA molecules of each of the target genes comprises determining the copy number of the mRNA molecules of each of the target genes. In some embodiments, determining the copy number of the mRNA molecules comprises reverse transcription of the mRNA molecules into cDNA molecules. In some embodiments, the cDNA molecules are quantified by digital PCR (dPCR). In some embodiments, the cDNA molecules are quantified by quantitative PCR (qPCR).

[0122] In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs). In some embodiments, the population of T cells is admixed with a population of non-T cells. In some embodiments, the non-T cells are tumor cells.

[0123] In some embodiments, the TILs are comprised in a tumor sample obtained from a patient. 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 mm3 being 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., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10mcg / 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.

[0124] In some embodiments, the method further comprises quantification of T cell DNA copy number of the population of T cells. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelically absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells. In some embodiments, the unique T cell DNA marker that is biallelically absent in T cells due to VDJ rearrangements is TRAB, the regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells is TRBC, and the reference DNA marker existing as a diploid in all cells is RPP30.

[0125] Experimental design governs the detection of the following DNA targets per assay: 1) AB as a unique T cell DNA marker that is biallelically absent in T cells due to VDJ rearrangements, 2) TRBC as a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and 3) RPP30 as a reference DNA marker existing as a diploid in all cells. The measurements of three markers are integrated to quantify the T cell DNA copy number / pL as follows:T cell quantification = (TRBC copy number -AB copy number)RPP30 copy number

[0126] After the assessment of tumor-reactivity of the population of TILs, the population of TILs may be further expanded using the methods disclosed herein.III. Method of Making TIL Product Enriched with NARTs

[0127] Some embodiments are directed to a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs).

[0128] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a. Generating a plurality of TIL populations by fractioning a tumor sample; b. Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); and c. Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate the TIL product enriched with NARTs.

[0129] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) selecting CD137+, CD200+ and / or CD103+ TILs from the first population of TILs to generate a second population of TILs; and e) Culturing the second population of TILs in a second cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

[0130] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) Culturing the first population of TILs in a second cell culture medium to generate a second population of TILs; and e) Culturing the second population of TILs in a third cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

[0131] In some embodiments, provided herein is a method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) Culturing the first population of TILs in a second cell culture medium to generate a second population of TILs; e) selecting CD137+, CD200+ and / or CD103+ TILs from the second population of TILs to generate a third population of TILs; and f) Culturing the third population of TILs in a third cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

[0132] Cell surface markers PD-1, CD39, and CD103 represent the leading biomarkers to identify tumor-specific T cells but these TILs are terminally differentiated. Up-regulation ofCD137 (4-1BB; TNFSR9) on recently activated CD8+ T-cells has been used to identify tumor antigen-specific T-cells (Draghi A., et al. Front Immunol 2021;12:705422, the content of which is hereby incorporated by reference in its entirety) which are antigen specific and tend to have a fitter / younger phenotype (c-fos and c-jun expression). CD137 through costimulation effects provides antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages.

[0133] Therefore, further provided is a method which enriches tumor-specific CD8 TILs (preserving the breadth of the TCR tumor-reactive repertoire), while providing CD4 TILs in the same process. In some embodiments, the method comprises priming tumors and use of CD137 as a marker to enrich and expand tumor-specific T cells without knowledge of epitope specificities. See, e.g., Fig. 1.A. Obtain Patient Tumor Sample

[0134] In general, TILs are initially obtained from a patient tumor sample ("primary TILs") and then expanded into a larger population for further manipulation as described herein.

[0135] A patient tumor sample may be obtained using methods known 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 (i.e., obtaining samples from one or more tumor cites 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 any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, renal, stomach, and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from endometrial cancer, cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breastcancer, triple negative breast cancer, and non-small cell lung carcinoma. In some embodiments, useful TILs are obtained from a melanoma.

[0136] 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 mm3 being 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., Roswell 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 of treating a cancer.B. Generating a Plurality of TIL Populations by Fractioning a Tumor Sample

[0137] In some embodiments, the methods provided herein comprise a step of generating a plurality of TIL populations by fractioning a tumor sample, for example, by fractioning the tumor into multiple tumor fragments, or by digesting the tumor into a tumor digest. In some embodiments, the tumor sample is fractioned into 2-1,000 tumor fragments. In some embodiments, the tumor sample is fractioned into 10-100 tumor fragments. In some embodiments, the tumor sample is fractioned into 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 tumor fragments.

[0138] 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 enzymatic digestion as whole tumors.In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and neutral protease. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and neutral protease for 1-2 hours. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and neutral protease 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 neutral protease 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 enzymes to form a tumor digest reaction mixture.

[0139] In some embodiments, the tumor is reconstituted with the lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0140] In some embodiments, the enzyme 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.

[0141] In some embodiments, the enzyme mixture comprises DNase. In some embodiments, the working stock for the DNase is a 10,000 lU / ml 10X working stock.

[0142] 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.

[0143] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 1000 lU / ml DNase, and 1 mg / ml hyaluronidase.

[0144] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 500 lU / ml DNase, and 1 mg / ml hyaluronidase.

[0145] In some embodiments, the enzyme mixture comprises neutral protease. In some embodiments, the working stock for the neutral protease is reconstituted at a concentration of 175 DMC U / mL.

[0146] In some embodiments, the enzyme mixture comprises neutral protease, DNase, and collagenase.

[0147] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 1000 lU / ml DNase, and 0.31 DMC U / ml neutral protease. In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 500 lU / ml DNase, and 0.31 DMC U / ml neutral protease.

[0148] In general, the harvested cell suspension is called a "primary cell population" or a "freshly harvested" cell population.

[0149] 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 patients.

[0150] In some embodiments, where the tumor is a solid tumor, the tumor undergoes physical fragmentation after the tumor sample is obtained. In some embodiments, the fragmentation occurs before 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 cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 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 mm3 to 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. In some embodiments, the multiple fragments comprise about to about 100 fragments.

[0151] 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 mm3 and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3 and 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.

[0152] 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 on each piece. In some embodiments, the tumors are resected in order to minimize the amount of fatty tissue on each piece.

[0153] 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 preforming 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 enzyme 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. Insome 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 contained a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

[0154] 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.

[0155] In some embodiments, cells can be optionally frozen after sample harvest and stored frozen prior to entry into the expansion described in further detail below.

[0156] In some embodiments, the TILs are obtained by thawing a cryopreserved tumor digest comprising a first population of TILs from a tumor resected from a subject. In some embodiments, the first population of TILs is obtained from a tumor resected from a subject by processing a tumor sample obtained from the subject into a tumor digest. In some embodiments, the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from the subject.

[0157] In some embodiments, the methods disclosed herein comprise adding a tumor digest or tumor fragments into a closed system, wherein the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from the subject. In some embodiments, the methods disclosed herein comprise performing a first expansion by thawing a cryopreserved tumor digest comprising a first population of TILs from a tumor that was resected from the subject. In some embodiments, the methods disclosed herein comprise obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into a tumor digest or multiple tumor fragments.

[0158] In some embodiments, a tumor lysate can be further obtained from the tumor digest through several freeze-thaw cycles or mass spectrometry procedures.

[0159] In some embodiments, the tumor fragments and / or tumor digest and / or tumor lysate can be optionally frozen and stored frozen prior to entry into the profiling step.

[0160] In some embodiments, the tumor is reconstituted with the lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0161] In some embodiments, the enzyme 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.

[0162] In some embodiments, the enzyme mixture comprises DNAse. In some embodiments, the working stock for the DNAse is a 10,000IU / mL 10X working stock.

[0163] 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.

[0164] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 lU / mL DNAse, and 1 mg / mL hyaluronidase.

[0165] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 ILI / mL DNAse, and 1 mg / mL hyaluronidase.

[0166] 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 patients. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.

[0167] In some embodiments, the TILs are not obtained from tumor digests. In some embodiments, the solid tumor cores are not fragmented.

[0168] In some embodiments, obtaining the first population of TILs comprises a multilesional sampling method.

[0169] 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 I (DNase), trypsin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof.

[0170] In some embodiments, the dissociating enzymes are reconstituted from lyophilized enzymes. In some embodiments, lyophilized enzymes are reconstituted in an amount of sterile buffer such as Hank's balance salt solution (HBSS).

[0171] In some instances, collagenase (such as animal free- type 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, about 280 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.

[0172] In some embodiments neutral protease is reconstituted in 1 mL 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 / mL, 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.

[0173] 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 to 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.

[0174] In some embodiments, the stock of enzymes could change so verify the concentration of the lyophilized stock and amend the final amount of enzyme added to the digest cocktail accordingly.

[0175] In some embodiments, the enzyme mixture includes about 10.2-ul of neutral protease (0.36 DMC U / mL), 21.3-ul of collagenase (1.2 PZ / mL) and 250-ul of DNase I (200 U / mL) in about 4.7 mL of sterile HBSS.C. Profiling the Plurality of TIL Populations

[0176] The invasion of tumors by tumor infiltrating lymphocytes (TILs) can impact patient outcomes and provide promising raw material for TIL manufacturing. Although flow cytometry and immunohistochemistry are considered gold-standard methods for T-cell quantification, these approaches are time consuming and require ample starting material. To circumvent these limitations, a multiplex digital PCR (dPCR) method (Nell et al., 2022 Methods in Molecular Biology, vol. 2453, 191-208; the content of which is herein incorporated by reference in its entirety) was developed for accurate and time-efficient T cell quantification using minimal starting material of tumor tissue.

[0177] Therefore, in some embodiments, the method disclosed herein comprises a profiling step, wherein the plurality of TIL populations comprised of tumor fragments or tumor digest is assessed for the T cell DNA copy number and / or the expression level of a panel of target genes using dPCR. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bia I lelica I ly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells. In some embodiments, quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bia I lelica lly absent in T cells due to VDJ rearrangements (e.g., AB). In some embodiments, quantification of T cell DNA copy number comprises detection of a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells (e.g., TRBC). In some embodiments, quantification of T cell DNA copy number comprises detection of a reference DNA marker existing as a diploid in all cells (e.g., RPP30). AB is a T cell-specific DNA sequence from the T cell receptor beta (TRB) gene, which is bia I lelica I ly absent in nearly all Tcells because it is deleted during V(D)J recombination. In contrast, AB is present on both alleles in all non-T cells, such as B cells, macrophages, or tissue cells, which retain the TRB gene in its unaltered germline configuration.

[0178] Expe rimental design governs the detection of the following DNA targets per assay: 1) AB as a unique T cell DNA marker that is bial lelica I ly absent in T cells due to VDJ rearrangements, 2) TRBC as a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and 3) RPP30 as a reference DNA marker existing as a diploid in all cells. The measurements of three markers are integrated to quantify the T cell DNA copy number / pL as follows:T cell quantification = (TRBC copy number -AB copy number)RPP30 copy number

[0179] Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates where adaptive anti-tumor cellular and humoral responses can be elaborated. Initially described in non-small cell lung cancer, TLS have also been found in melanomas and sarcomas, and associated with improved response to immunotherapy. TLS are composed predominantly of aggregates of B cells and T cells, follicular dendritic cells (FDCs) and may have a role in the improved survival of patients.

[0180] Therefore, to assess the tumor reactivity of TILs, a genetic panel of TLS markers that are overexpressed in tumor reactive TILs are designed using transcriptomic data. In some embodiments, the genetic panel comprises one or more genes selected from the list including CXCL13, CD200, TRBC, YWHAZ, CD19, CD20, etc.

[0181] In some embodiments, profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations. In some embodiments, the panel of target genes comprises one or more genes selected from CXCL13, CD200, TRBC, YWHAZ, CD20, and CD19. In some embodiments, the panel of target genes includes CXCL13. In some embodiments, the panel of target genes includes CD200. In some embodiments, the panel of target genes includes TRBC. In some embodiments, the panel of target genes includes YWHAZ. In some embodiments, the panelof target genes includes CD20. In some embodiments, the panel of target genes includes CD19.

[0182] In some embodiments, the method disclosed herein comprises selecting one or more TIL populations enriched with NARTs based on the results from the profiling step, wherein the plurality of TIL populations comprised of tumor fragments or tumor digest is assessed for the T cell DNA copy number and / or the expression level of a panel of target genes using dPCR. In some embodiments, one or more TIL populations, such as tumor fragments, that exhibit the highest T cell DNA copy number and / or the highest expression level of the panel of target genes will be selected for further expansion and / or culturing steps disclosed herein.D. Preferential Expansion of NARTs

[0183] In some embodiments, the method disclosed herein comprises a preferential expansion step, wherein the one or more TIL populations enriched with NARTs are cocultured with tumor cells from the tumor sample in a first cell culture medium. In some embodiments, the tumor cells are from a tumor digest of the tumor sample.

[0184] In some embodiments, the first cell culture medium IFNy. In some embodiments, the IFNy is present at a concentration of about 10 ng / mL to about 1000 ng / mL, e.g., about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 100 ng / mL, about 200 ng / mL, about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1,000 ng / mL. In some embodiments, the IFNy is present at a concentration of about 100 ng / mL. In some embodiments, the IFNy is present at a concentration of about 200 ng / mL. In some embodiments, the IFNy is present at a concentration of about 400 ng / mL.

[0185] In some embodiments, the first cell culture medium comprises an immune checkpoint inhibitor (ICI). In some embodiments, the ICI comprises a CD38 inhibitor, a CTLA- 4 inhibitor and / or a PD-1 / PD-L1 inhibitor. In some embodiments, the first cell culture medium comprises soluble IL-12 and / or a 4-1BB agonist. In some embodiments, the 4-1BB agonist is urelumab.

[0186] In some embodiments, the first cell culture medium comprises a combination of two or more of IL-2, IL-15 and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, 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.

[0187] In some embodiments, the first cell culture medium comprises IL-2. In some embodiments, the IL-2 is at a concentration of 3000 ID / mL or lower. In some embodiments, the first cell culture medium comprises no added IL-2. In some embodiments, the first cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, the first cell culture medium comprises IL-15 and / or IL- 21 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises IL-2 at 3000 ID / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium comprises IL-15 and IL-21. In some embodiments, the first cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

[0188] The preferential expansion step may last for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 48 hours. In some embodiments, the preferential expansion step lasts for 12 hours. In some embodiments, the preferential expansion step lasts for 24 hours. In some embodiments, the preferential expansion step lasts for 30 hours. In some embodiments, the preferential expansion step lasts for 36 hours. In some embodiments, the preferential expansion step lasts for 48 hours.E. Selecting Tumor Reactive TILs

[0189] In some embodiments, the population of TILs after the preferential expansion step, and / or the first expansion (pre-REP) step is selected for CD137+ TILs. In some embodiments, the TIL population after the preferential expansion step, and / or the first expansion (pre-REP) step disclosed herein is positively selected for tumor reactive TILs. By way of example, a cell that is "CD137+" or that "expresses CD137" is contrasted herein to a cell that is CD137- ordoes not express a detectable level of CD137. A cell that is "CD137dim," as used herein has a lower detectable level of CD137 expression than a CD137+ cell or a "CD137bright" cell. In one embodiment, the tumor-reactive T cells are positive for both CD137 and PD-1. By way of example, a cell that is "CD137+PD-1+" or that "expresses CD137 and PD-1" is contrasted herein to a cell that is CD137-PD-1+, CD137+PD-1- or does not express a detectable level of either CD137 or PD-1.

[0190] The present disclosure encompasses a method for selecting CD137+ cells using a straightforward direct antibody-based purification system for isolating such cells. However, any cellular isolation methodology can be used to isolate the cells from a biological sample. For example, an antibody that binds to CD137 can be bound to a physical support, such as a magnetic bead, a Dynal bead, a microbead, a column, an adsorption column, and an adsorption membrane. Conjugating an antibody to a physical support is well known in the art. Alternatively, an antibody conjugated to a physical support, such as a magnetic bead, can be purchased from a variety of sources, such as Milteny Biotec (Auburn, Calif.).

[0191] Literature shows that some tumor-reactive TILs reside in the CD137+ population. CD39 is more selective and is expressed by the majority of the putative CD4 tumor reactive (PD1+ICOS+) population. CD200 is expressed by the majority of the putative tumor reactive CD4+CXCL13 population. Therefore, in some embodiments, the selecting step may comprise selecting TILs that express CD200 and / or CD39. Further, 0X40 is a known activation marker for CD4 T cells that would potentially be upregulated 24 hours after TCR stimulation in digest culture. Therefore, in some embodiments, the selecting step may comprise selecting TILs that express CD200, CD39 and / or 0X40.

[0192] By including an anti-CD200 antibody, an anti-C39 antibody and / or an anti-OX40 antibody in addition to an anti-CD137 antibody, the yield of tumor reactive TILs will be increased by the method disclosed herein, which still removing a very significant fraction of the bystander TILs. In some embodiments, the tumor reactive TILs may be a population of CD200+ cells. In some embodiments, the tumor reactive TILs may be a population of CD39+ ce I Is. In some embodiments, the tumor reactive TILs may be a population of 0X40+ cells.

[0193] In some embodiments, enriching the population of TILs for CD137+ TILs comprises contacting the population of TILs with anti-CD137 antibody immobilized on a bead. In someembodiments, the method further comprises contacting the population of TILs with an anti-CD39 antibody immobilized on a bead. In some embodiments, the method further comprises contacting the population of TILs with an anti-CD200 antibody immobilized on a bead. In some embodiments, the method further comprises contacting the population of TILs with an anti-OX40 antibody immobilized on a bead.

[0194] A variety of antibodies may be useful in the methods described herein. As will be understood by one skilled in the art, any antibody that can recognize and bind to an antigen of interest, such as CD137, CD200, CD39, or 0X40, is useful in the methods. Methods of making and using such antibodies are well known in the art. For example, polyclonal antibodies are generated by immunizing rabbits according to standard immunological techniques well-known in the art.

[0195] In some embodiments, the method further comprises removing PD-lHi TILs from the TIL product enriched with NARTs or the positively-selected TIL product to generate a bystander-depleted TIL product.F. First Expansion (Pre-REP)

[0196] In some embodiments, the tumor reactive TILs resulting from the preferential expansion step, or from the selecting step are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. This expansion is referred to herein as the first expansion, which can include expansion processes generally referred to in the art as a pre-rapid expansion process (pre-REP). In some embodiments, the tumor reactive TILs resulting from the preferential expansion step, or the selecting step are incubated (e.g., in 2 mL wells) in media comprising inactivated human AB serum with 6000 lU / mL of IL-2. In some embodiments, the tumor reactive TILs are cultured for a period of days, generally from 3 to 14 days. In some embodiments, the tumor reactive TILs are cultured for a period of 5 to 11 days. In some embodiments, the tumor reactive TILs are cultured for a period of 7 to 10 days. In some embodiments, the tumor reactive TILs are cultured for a period of about 9 days.

[0197] In embodiments where TIL cultures are initiated in 24-well plates, for example, using Costar 24-well cell culture cluster, flat bottom (Corning Incorporated, Corning, NY),each well can be seeded with 1 x 106 tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 lU / mL; Chiron Corp., Emeryville, CA).

[0198] In some embodiments, the first expansion culture medium is referred to as "CM", an abbreviation for culture media. In some embodiments, CM for the first expansion 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 cm2 gas-permeable silicon bottom (for example, G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN, each flask may be loaded with 10-40 x 106 viable 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 may be incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media may be removed and replaced with fresh CM and IL-2 and after day 5, half the media may be changed every 2-3 days.

[0199] In some embodiments, the tumor reactive TILs resulting from the preferential expansion step, or the selecting step are cultured in a second cell culture medium comprising feeder cells. In some embodiments, the feeder cells are T cell-depleted feeder cells, such as T cell-depleted PBMCs (see Fig. 1).

[0200] 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 serum-containing media.

[0201] 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.

[0202] 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 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+, AI3+, 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.

[0203] 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.

[0204] 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.

[0205] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ can 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.

[0206] 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 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 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, theCTS™OpTmizer™ T-cell Expansion SFM is supplemented with about 3% of the CTS™ ImmuneCell 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 ID / 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.

[0207] 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 mM, 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 (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0208] In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5mM to about 150mM, lOmM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about llOmM, 30mM to about lOOmM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the media is 55pM.

[0209] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, are used. 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 aserum-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 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 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 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+, AI3+, 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 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.

[0210] In some embodiments, the concentration of glycine in the defined medium is in the range of from about 5 to about 200 mg / L, the concentration of L- histidine is about 5 to about 250 mg / L, the concentration of L-isoleucine is about 5 to about 300 mg / L, the concentration of L-methionine is about 5 to about 200 mg / L, the concentration of L- phenylalanine is about 5 to about 400 mg / L, the concentration of L-proline is about 1 to about 1000 mg / L, the concentration of L- hydroxyproline is about 1 to about 45 mg / L, the concentration of L-serine is about 1 to about 250 mg / L, the concentration of L-threonine is about 10 to about 500 mg / L, the concentration of L-tryptophan is about 2 to about 110 mg / L, the concentration of L-tyrosine is about 3 to about 175 mg / L, the concentration of L-valine is about 5 to about 500 mg / L, the concentration of thiamine is about 1 to about 20 mg / L, the concentration of reduced glutathione is about 1 to about 20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1 to about 200 mg / L, the concentration of iron saturated transferrin is about 1 to about 50 mg / L, the concentration of insulin is about 1 to about 100 mg / L, the concentration of sodium selenite is about 0.000001 to about 0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5000 to about 50,000 mg / L.

[0211] 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 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 below.TABLE 1: Concentrations of Non-Trace Element Moiety Ingredients

[0212] 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).

[0213] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038 / cti.2014.31) are used. 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.

[0214] 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 firstand / or second gas permeable container lacks beta-mercaptoethanol (BME or PME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0215] After preparation of the tumor fragments, the resulting cells (i.e., fragments) 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 (or, in some cases, in the presence of aAPC 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 1x108 bulk 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 (rhlL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30x106 lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20x106 lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25x106 lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30x106 lU / mg for a 1 mg vial. In some embodiments, the IL- 2 stock solution has a final concentration of 4-8x106 lU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 5-7x106 lU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 6x106 lU / mg of IL-2. 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 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, about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about4000 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, between 1000 and 5000 lU / mL, or about 8000 lU / mL of IL-2.

[0216] In some embodiments, the first expansion can proceed for about 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 expansion can proceed for 1 day to 14 days. In some embodiments, the first expansion can proceed for 2 days to 14 days. In some embodiments, the first expansion can proceed for 3 days to 14 days. In some embodiments, the first expansion can proceed for 4 days to 14 days. In some embodiments, the first expansion can proceed for 5 days to 14 days. In some embodiments, the first expansion can proceed for 6 days to 14 days. In some embodiments, the first expansion can proceed for 7 days to 14 days. In some embodiments, the first expansion can proceed for 8 days to 14 days. In some embodiments, the first expansion can proceed for 9 days to 14 days. In some embodiments, the first expansion can proceed for 10 days to 14 days. In some embodiments, the first expansion can proceed for 11 days to 14 days. In some embodiments, the first expansion can proceed for 12 days to 14 days. In some embodiments, the first expansion can proceed for 13 days to 14 days. In some embodiments, the first expansion can proceed for 14 days. In some embodiments, the first expansion can proceed for 1 day to 11 days. In some embodiments, the first expansion can proceed for 2 days to 11 days. In some embodiments, the first expansion can proceed for 3 days to 11 days. In some embodiments, the first expansion can proceed for 4 days to 11 days. In some embodiments, the first expansion can proceed for 5 days to 11 days. In some embodiments, the first expansion can proceed for 6 days to 11 days. In some embodiments, the first expansion can proceed for 7 days to 11 days. In some embodiments, the first expansion can proceed for 8 days to 11 days. In some embodiments, the first expansion can proceed for 9 days to 11 days. In some embodiments, the first expansion can proceed for 10 days to 11 days. In some embodiments, the first expansion can proceed for about 11 days. In some embodiments, the first expansion can proceed for 5 days to 7 days. In some embodiments, the first expansion can proceed for 6days to 7 days. In some embodiments, the first expansion can proceed for 7 days to 12 days. In some embodiments, the first expansion can proceed for 8 days to 12 days. In some embodiments, the first expansion can proceed for 9 days to 12 days. In some embodiments, the first expansion can proceed for 10 days to 12 days. In some embodiments, the first expansion can proceed for about 7 days. In some embodiments, the first expansion can proceed for about 9 days.

[0217] In some embodiments, the first expansion 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.

[0218] In some embodiments, the first cell culture medium comprises 6000 ILI / mL of IL-2. In some embodiments, the first cell culture medium comprises 3000 lU / mL of IL-2. In some embodiments, the first cell culture medium comprises 2000 lU / mL of IL-2. In some embodiments, the first cell culture medium comprises 1000 lU / mL of IL-2.G. Second Expansion (REP)

[0219] In some embodiments, the TIL cell population is expanded in number after initial bulk processing, profiling, preferential expansion of NARTs, selecting tumor reactive TILs, and / or pre-REP expansion. 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). 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 agonist antibody, in a gas-permeable container.

[0220] In some embodiments, the second expansion (which can include expansions sometimes referred to as REP) of TIL can be performed using any TIL flasks or containers known by those of skill in the art. In some embodiments, the second 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 expansion can proceed for about 7 days to about 14 days. In some embodiments, the second expansion can proceed for about 7 days to about 12 days. In some embodiments, the second expansion can proceed for about 7 days to about 10 days. In someembodiments, the second expansion can proceed for about 7 days to about 9 days. In some embodiments, the second expansion can proceed for about 8 days to about 9 days. In some embodiments, the second expansion can proceed for about 9 days. In some embodiments, the second expansion can proceed for about 10 days. In some embodiments, the second expansion can proceed for about 11 days.

[0221] 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). 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 nonspecific T-cell receptor stimulus can include, for example, an anti-CD3 agonist antibody, such as OKT3 (e.g., about 30 ng / ml 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 growth 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.

[0222] 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, about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000I U / m L, about 4500 lU / mL, about 5000 I U / m L, 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.

[0223] 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, or 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.

[0224] 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.

[0225] 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, 30mg / mL OKT3 anti-CD3 antibody and 3000 lU / 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 growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.

[0226] In some embodiments, the second expansion (which can include processes referred to as the REP process) is shortened to 7-11 days, as discussed in the examples and figures. In some embodiments, the second expansion is shortened to 9 days. In some embodiments, the second expansion is shortened to 8 days. In some embodiments, the second expansion is shortened to 7 days. In some embodiments, the second expansion is shortened to 6 days. In some embodiments, the second expansion is shortened to 5 days. In some embodiments, the second expansion is shortened to 4 days.

[0227] In some embodiments, REP and / or the second expansion may be performed using T-175 flasks and gas permeable bags as previously described (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 106 TILs 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 ID 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 III 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 III 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 106 cells / mL.

[0228] In some embodiments, the second expansion (which can include expansions referred to as REP) 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 106 or 10 x 106 TIL 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 beincubated at 37°C in 5% C02. 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 ID 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 III per mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C in 5% CO2 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.

[0229] In some embodiments, the second expansion (which can include expansions referred to as REP) 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 106 or 10 x 106 TIL 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 (or G-REX100M) 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, 6000 IU per mL of IL-2, and added back to the original GREX-100 flasks. When TILs are expanded serially in GREX-100 flasks, on day 10 or 11 the TILs can be moved to a larger flask, such as a GREX-500 (or G-REX500M). The cells may be harvested on day 14 of culture. The cells may be harvested on day 15 of culture. The cells may be harvested on day 16 of culture. 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 aspiration of spent media and replacement with an equal volume of fresh media. In some embodiments, alternative growth chambers include GREX flasks and gas permeable containers as more fully discussed below. In some embodiments, the process employed varying centrifugation speeds (400g, 300g, 200g for 5 minutes) and varying numbers of repetitions.

[0230] 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 aspiration of spent media followed by infusion with fresh media. In some embodiments, alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.

[0231] 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.

[0232] 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 serum-containing media.

[0233] 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.

[0234] 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 collagenprecursors, 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 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+, AI3+, 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.

[0235] 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.

[0236] 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.

[0237] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present methods. 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 CellSerum 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.

[0238] 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 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 1000 ILI / 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, theCTS™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 ILI / mL to about 8000 lU / mL of IL-2. In someembodiments, 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 IlJ / 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.

[0239] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of from about O.lmM to about lOmM, 0.5mM to about 9mM, ImM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2mM.

[0240] In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5mM to about 150mM, lOmM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about llOmM, 30mM to about lOOmM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the media is 55pM.

[0241] In some embodiments, the defined media described in International PCT Publication No. WO / 1998 / 030679, which is herein incorporated by reference, are used. 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 one or more albumins or albuminsubstitutes, 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 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 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+, AI3+, 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 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.

[0242] 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 about0.000001-0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5000-50,000 mg / L.

[0243] 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 2 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 2. 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 2 below.TABLE 2: Concentrations of Non-Trace Element Moiety Ingredients

[0244] 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).

[0245] In some embodiments, the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038 / cti.2014.31) are used. 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.

[0246] 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).

[0247] In some embodiments, the second expansion 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 someembodiments, 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.

[0248] In some embodiments, the steps of the method are completed within a period of about 22 days. In some embodiments, the steps of the method are completed within a period of about 8 days. In some embodiments, the steps of the method are completed within a period of about 9 days. In some embodiments, the steps of the method are completed within a period of about 10 days. In some embodiments, the steps of the method are completed within a period of about 11 days. In some embodiments, the steps of the method are completed within a period of about 12 days. In some embodiments, the steps of the method are completed within a period of about 13 days. In some embodiments, the steps of the method are completed within a period of about 14 days. In some embodiments, the steps of the method are completed within a period of about 15 days. In some embodiments, the steps of the method are completed within a period of about 16 days. In some embodiments, the steps of the method are completed within a period of about 17 days. In some embodiments, the steps of the method are completed within a period of about 18 days. In some embodiments, the steps of the method are completed within a period of about 19 days. In some embodiments, the steps of the method are completed within a period of about 20 days. In some embodiments, the steps of the method are completed within a period of about 21 days. In some embodiments, the steps of the method are completed within a period of about 22 days. In some embodiments, the steps of the method are completed within a period of about 23 days. In some embodiments, the steps of the method are completed within a period of about 24 days. In some embodiments, the steps of the method are completed within a period of about 25 days. In some embodiments, the steps of the method are completed within a period of about 26 days. In some embodiments, the steps of the method are completed within a period of about 27 days. In some embodiments, the steps of the method are completed within a period of about 28 days. In some embodiments, the steps of the method are completed within a period of about 29 days. In some embodiments, the steps of the method are completed within a period of about 30 days. In some embodiments, the steps of the method are completed within a period of about 31 days.

[0249] In some embodiments, the antigen presenting cells (APCs) are PBMCs. According to some embodiments, the PBMCs are irradiated. According to some embodiments, the PBMCs are allogeneic. According to some embodiments, the PBMCs are irradiated and allogeneic. According to some embodiments, the antigen-presenting cells are artificial antigen- presenting cells.

[0250] In some embodiments, the IL-2 is present at an initial concentration of between 1000 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4000 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4500 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 5000 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 5500 lU / mL and 6000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 lU / mL and 5000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 lU / mL and 5000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 lU / mL and 5000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 lU / mL and 5000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 lU / mL and 5000 lU / mL in the cellculture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 ILI / mL and 5000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4000 lU / mL and 5000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4500 ILJ / mL and 5000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 lU / mL and 4000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 lU / mL and 4000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 lU / mL and 4000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 lU / mL and 4000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 lU / mL and 4000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 lU / mL and 4000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 lU / mL and 3000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 ILI / mL and 3000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 lU / mL and 3000 ILI / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 lU / mL and 3000 lU / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 lU / mL and 2000 ILI / mL in the cell culture medium in the first expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 lU / mL and 2000 lU / mL in the cell culture medium in the first expansion.

[0251] In some embodiments, the second expansion step, the IL-2 is present at an initial concentration of between 1000 lU / mL and 6000 lU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0252] In some embodiments, the first expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container.

[0253] In some embodiments, the first cell culture medium further comprises a cytokine selected from IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and / or third culture medium further comprises a cytokine selected from IL-4, IL-7, IL-15, IL-21, and combinations thereof.1. Feeder Cells and Antigen Presenting Cells

[0254] In some embodiments, the second expansion procedures described herein 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.

[0255] 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 an exemplary protocol for evaluating the replication incompetence of irradiate allogeneic PBMCs.

[0256] 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).

[0257] 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 IU / mL IL-2.

[0258] 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 ID / rnL 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.

[0259] 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.

[0260] In some embodiments, the second expansion procedures described herein require a ratio of about 2.5x109 feeder cells to about 100x106 TIL. In other embodiments, the second expansion procedures described herein require a ratio of about 2.5x109 feeder cells to about 50x106 TIL. In yet other embodiments, the second expansion procedures described herein require about 2.5x109 feeder cells to about 25x106 TIL.

[0261] 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 asFicoll-Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0262] In some embodiments, artificial antigen presenting cells (aAPC) are used in the second expansion as a replacement for, or in combination with, PBMCs. In some embodiments, the aAPCs are acellular-based or cellular-based.

[0263] In some embodiments, the artificial antigen presenting cell (aAPC) comprises a U937 cell expressing one or more co-stimulatory molecules. In some embodiments, the one or more co-stimulatory molecules are selected from: a CD64 protein, a CD86 protein, a 4- 1BBL protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-1BBL protein, and an OX40L protein. In some embodiments, the aAPC can stimulate and expand a T cell contacted with the aAPC. In some embodiments, the aAPC can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the aAPC. In some embodiments, the aAPC expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 lU / mL and OKT-3 antibody at a concentration of about 30 ng / mL.

[0264] In some embodiments, the artificial antigen presenting cell (aAPC) comprises a lipid nanoparticle (LNP) comprising one or more co-stimulatory molecules. In some embodiments, the one or more co-stimulatory molecules are selected from: a CD64 protein, a CD86 protein, a 4-1BBL protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a CD86 protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and a 4-1BBL protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, and an OX40L protein. In some embodiments, the one or more co-stimulatory molecules comprise a CD64 protein, a CD86 protein, a 4-1BBL protein, and an OX40L protein. In some embodiments, the LNP can stimulate and expand a T cell contacted with the LNP. In some embodiments, the LNP can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the LNP. In some embodiments, the LNP expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 lU / mL and OKT-3 antibody at a concentration of about 30 ng / mL.

[0265] In some embodiments, the LNP comprises: (a) at least one ionizable lipid; (b) at least one neutral lipid; (c) cholesterol and / or a modified derivative thereof; and (d) at least one polymer conjugated lipid and / or a modified derivative thereof. In some embodiments, the one or more co-stimulatory molecules are covalently conjugated to at least one component of the LNP. In some embodiments, the component to which the one or more co- stimulatory molecules are covalently conjugated is the modified derivative of the polymer conjugated lipid. In some embodiments, the covalent conjugation comprises a covalent bond forming reaction selected from a [l,4]-conjugate addition (i.e., Michael addition), [4+2] cycloaddition, [3+2] dipolar cycloaddition, nucleophilic addition, transition metal-catalyzed cross-coupling reaction, carbonyl condensation reaction, and reductive amination. In some embodiments, the covalent conjugation reaction comprises a [l,4]-conjugate addition reaction (i.e., Michael addition). In some embodiments, the [l,4]-conjugate addition occurs between the modified derivative of the polymer conjugated lipid which is further conjugated to a maleimide moiety and a cysteine thiol of a polypeptide. In some embodiments, the cystine thiol of the polypeptide is derived from a reduced disulfide bridge of the one or more co-stimulatory molecules.2. Cytokines and Other Additives

[0266] 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.

[0267] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is 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.

[0268] In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 is at a concentration of 3000 lU / mL or lower. In some embodiments, the first cell culture medium or the second cell culture medium contains no added IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises IL-2 at 3000 lU / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol.

[0269] In some embodiments, the first expansion is performed over a period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 7-11 days.3. Modifiers of T Cell Metabolism

[0270] In vitro expansion alters TIL cellular state, which correlates with ACT efficacy (Chiffe lie, J., et al., bioRxiv 2023, the content of which is hereby incorporated by reference in its entirety). Rapidly expanding TILs have bioenergetic and biosynthetic needs. Increasing the availability of essential cofactors such as L-arginine and NAD+ may support synthesis of major biomass components. To functionally reinvigorate cells during in vitro expansion may produce TILs with renewed effector features.

[0271] Following the selection of tumor-specific TIL, TR TIL clones may be cultured in conditions that simulate the nutrient restricted tumor microenvironment in hypoxia (5% molecular oxygen) in low glucose (1.5-3 mM) supplemented with molecules that serve as an alternative carbon and energy to meet the bioenergetic and biosynthetic needs of dividing TR TIL that includes but not restricted to Na-L-lactate (pH neutral form, 0.5-5mM), short chain fatty acids Beta-hydroxy butyrate, p-OHB (l-5mM) or Sodium butyrate (0.5mM), Sodium Propionate 0.1-lmM, Sodium Acetate 1-lOmM. Expansion conditions also include but not restricted to mitophagy activators metformin, valproic acid, Urolithin A together with NAD+ boosters to promote mitochondrial fitness by selectively targeting damaged mitochondria for degradation.

[0272] Therefore, provided herein are modifiers of T cell metabolism that increase availability of essential cofactors L-arginine and NAD+to support synthesis of major biomass components in TILs, which may be added to the second expansion (REP) of TILs.

[0273] L-arginine, a building block for protein synthesis, improves T cell function via metabolic modification (Geiger R., et al., Cell 2016: 167; 829-842; Fultang, L., Blood 2020; 136: 1155-60; the contents of which are hereby incorporated by reference in their entireties).

[0274] NAD+ (nicotinamide adenine dinucleotide), an electron acceptor, is heavily consumed during biomass synthesis, and supplementation improves T cell function (Canto C, et al., Cell Metabolism 2015; 22:31-53; Wang Y, et al., Cell Reports 2021; 36:1-12; the contents of which are hereby incorporated by reference in their entireties).

[0275] NAD+ (Nicotinamide adenine dinucleotide) is a central coenzyme in cellular metabolism, particularly in redox reactions where it cycles between oxidized (NAD+) and reduced (NADH) states. In the context of TILs, NAD+ plays a critical role in modulating their metabolic fitness and function. TILs operate in the tumor microenvironment, which is often characterized by nutrient scarcity and hypoxia. In this challenging microenvironment, TILs must adapt their metabolism to sustain their anti-tumor activity.

[0276] NAD+ enhances the metabolic fitness of T cells in several ways:

[0277] Glycolysis and the Pentose Phosphate Pathway (PPP): Although glycolysis does not directly use NAD+, the regeneration of NAD+ from NADH is critical to maintain glycolytic flux, which is particularly important in hypoxic conditions where mitochondrial respiration is limited. The PPP, which branches from glycolysis, relies on NADP+ (the phosphorylated form of NAD+) for the generation of ribose-5-phosphate and for the reduction of glutathione, contributing to nucleotide synthesis and antioxidant defense mechanisms.

[0278] Mitochondrial Function: NAD+ is essential in the mitochondria as it is a key electron acceptor in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS).Enhanced mitochondrial function can support the differentiation and survival of memory T cells, which are crucial for sustained anti-tumor immunity.

[0279] S irtuins Activation: Sirtuins are a family of NAD-r-dependent deacetylases that regulate cellular metabolism, stress responses, and inflammation. By activating sirtuins, NAD+ can promote the formation of memory T cells and enhance their anti-tumor function.

[0280] PARP Activity: NAD+ is a substrate for poly(ADP-ribose) polymerases (PARPs) which are involved in DNA repair and genomic stability. By facilitating the function of PARPs, NAD+ helps maintain TIL integrity in the face of DNA-damaging conditions within tumors.

[0281] To replicate and enhance the benefits provided by NAD+ to TILs, both pharmacological and genetic approaches can be considered:

[0282] Pharmacologic Approaches:NAD+ Precursors: Boosting NAD+ via administration of NAD+ precursors such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) can increase intracellular NAD+ levels and thereby support TIL metabolism and function.NAD+: Boosting availability of NAD+ metabolic substrate directly to TILs Sirtuin Activators: Compounds like resveratrol or SRT1720 can activate sirtuins, thus mimicking the effect of high NAD+ levels and promoting TIL anti-tumor activity. PARP Inhibitors: While PARP activity is NAD+ dependent and can be beneficial, excessive activation can deplete cellular NAD+ reserves. PARP inhibitors can help preserve NAD+ levels under stress conditions, potentially supporting T cell function. CD38 Inhibitors: CD38 uses NAD+ to generate ADP-ribose and cyclic ADP-ribose, which are important for calcium signaling but also deplete NAD+. Inhibitors of CD38 may help preserve NAD+ levels in TILs.

[0283] Genetic Approaches:Overexpression of NAMPT: This is the rate-limiting enzyme in the NAD+ salvage pathway. Overexpressing NAMPT can boost the intracellular levels of NAD+ in TILs. Manipulation of Sirtuin Genes: Genetic modification to overexpress sirtuin genes can potentially mimic the effect of high NAD+ levels and enhance TIL function.Gene Silencing of NAD+ Consuming Enzymes: Silencing or knocking down genes encoding for enzymes that heavily consume NAD+, like CD38 or PARP family members, might conserve NAD+ for metabolic processes essential for TILs. TCA Cycle and OXPHOS Support: Enhancing genes involved in mitochondrial biogenesis and function can support the efficient use of NAD+ and maintain TIL metabolic fitness.

[0284] Pharmacologic and genetic tools to augment NAD+ utilization in TILs center on specific molecular targets and pathways. In some embodiments, exemplary tools for augment NAD+ utilization focus on one or more of the following functions: mitochondrial biogenesis, promoting effective energy utilization, and fostering an anti-tumor phenotype. In embodiments, such tools are implemented in a concerted manner, with attention to the balance between enhancing TIL function and avoiding over-stimulation that could lead to exhaustion or apoptosis. In some embodiments, the heterogeneity within the TIL population and the unique microenvironment of each tumor when designing these interventions are taken into consideration. The modulation of mitochondrial function through pharmacological and genetic approaches could thus be a powerful strategy to enhance the efficacy of adoptive TIL therapies. Tools for augmenting NAD+ utilization are further detailedbelow. One or more of the following tools, pathways, or modulators may be used in the methods described herein. Additional embodiments are provided in the table.Pharmacological Manipulation of NAD+ Pathways:1. NAD+ Precursors and NAD+: o Nicotinamide Riboside (NR) and Nicotinamide Mononucleotide (NMN) are NAD+ precursors. They are converted to NAD+ via the salvage pathway involving the enzyme nicotinamide riboside kinase (NRK1 / 2) for NR and NMN adenylyltransferase (NMNAT1 / 2 / 3) for NMN. o Nicotinic Acid (NA) can also be used, which is converted into NAD+ via the Preiss-Handler pathway, although it can cause flushing due to prostaglandin- mediated vasodilation. o Nicotinamide dinucleotide (NAD+) itself can also be used, as it has been shown to cross the lipid bilayer and enter the mitochondria.2. Sirtuin Modulators: o Resveratrol is a si rtu in-activating compound (STAC) that enhances SIRT1 activity, which in turn can deacetylate and activate peroxisome proliferator- activated receptor gamma coactivator 1-alpha (PGC-la), a master regulator of mitochondrial biogenesis. o SRT1720, another STAC, has been shown to selectively activate SIRT1, leading to enhanced mitochondrial function and possible improvement in TIL survival and function.3. PARP Inhibitors: o Compounds like Olaparib and Niraparib can inhibit PARP enzymes, thus conserving NAD+ for other metabolic processes critical for T cell function and potentially reducing the metabolic competition between PARPs and SIRT1 for NAD+.4. CD38 Inhibitors:o Isatuximab and Daratumumab are monoclonal antibodies that target CD38, which is responsible for NAD+ degradation. Small molecule inhibitors of CD38 are also in development and could be repurposed to enhance NAD+ levels in TILs.5. Mitochondrial Metabolic Modulators: o Metformin has been shown to activate AMP-activated protein kinase (AMPK), which in turn activates PGC-la, improving mitochondrial biogenesis and potentially restoring TIL function. o Pioglitazone, a PPARy agonist, might also enhance mitochondrial biogenesis and function in TILs through PGC-la induction. o Bezafibrate: A pan-PPAR agonist that can induce PGC-la, enhancing mitochondrial function and fatty acid oxidationMitochondrial Biogenesis and Dynamics:1. PGC-la Activation: o Specific activators of PGC-la or direct overexpression via gene delivery systems could be used to stimulate mitochondrial biogenesis.2. Mitochondrial Dynamics: o Manipulating genes involved in mitochondrial fusion (e.g., MFN1 / 2, 0PA1) and fission (e.g., DRP1, FIS1) to maintain mitochondrial network integrity and function.Genetic Tools:1. Overexpression of NAD+ Synthesis Genes: o NAMPT: Overexpression can increase the salvage pathway flux, boosting NAD+ levels. o NMNAT1 / 2 / 3: Increased expression of NMN adenylyltransferases to catalyze the conversion of NMN to NAD+.Gene Manipulation: SIRT1-7: Overexpressing Sirtuin genes, with a focus on SIRT1 for its role in mitochondrial biogenesis, SIRT3 for mitochondrial protein deacetylation, and SIRT6 for maintaining genomic stability. lation of TCA Cycle and OXPHOS Genes: PC (Pyruvate Carboxylase): Enhancing anaplerotic reactions to replenish TCA cycle intermediates. PDK (Pyruvate Dehydrogenase Kinase) Inhibitors: Genetic silencing of PDK to increase PDH activity and promote glucose oxidation. gulation of NAD+ Consuming Enzymes: Using CRISPR / Cas9 or siRNA approaches to knockdown CD38 or PARP family members to conserve NAD+ for enhancing TIL function. ion of Mitochondrial DNA Stability and Biogenesis: TFAM (Mitochondrial Transcription Factor A): Overexpression can promote mitochondrial DNA replication and transcription, thus enhancing mitochondrial function. OGGI or MUTY : Overexpression to enhance base-excision repair and maintain mitochondrial DNA integrity. alance: GCLC / GCLM (Glutamate-Cysteine Ligase Catalytic and Modifier Subunit): Overexpression to increase glutathione synthesis, a key antioxidant within mitochondria. lic Reprogramming: CPT1A (Carnitine Palmitoyltransferase 1A): Genetic modification to boost fatty acid oxidation, which could sustain TIL function under nutrient-deprived conditions.8. Mitophagy and Autophagy Regulators: o PRKN (Parkin) and PINK1: Enhance mitophagy to remove dysfunctional mitochondria. o ATG5 or ATG7: Modulation of autophagy-related genes to balance energy and organelle quality control in TILs.

[0285] In some embodiments, these strategies may be optimized for dose, timing, and delivery mechanisms to ensure the TILs are metabolically enhanced without inducing detrimental effects such as apoptosis or senescence. In embodiments, such strategies are also assessed to avoid the promotion of tumor cell survival or immune evasion. In some embodiments, a combination of these strategies, informed by the specific metabolic deficiencies observed in the TILs isolated from individual tumors, provides a potent and personalized enhancement of TIL function for adoptive cell therapies.

[0286] The evolutionary conserved Wnt / β -catenin pathway is critically involved in pluripotency and differentiation of embryonic stem cells via maintenance of DNA methylation patterns promoting epigenetic stability necessary for correct cell fate decisions during states of high cell division (Clevers, H. and R. Nusse, Cell, 2012. 149(6): p. 1192-205). In humans, rapid recall of memory T cells upon re-exposure to antigen suggests the requirement of a stable epigenetic program. Most TIL-based ACT protocols for solid tumors are generated from terminally differentiated effector memory T cells resulting from repetitive rounds of TCR ligation during the ex vivo phase (Hinrichs, C.S. and S.A. Rosenberg, Immunol Rev, 2014. 257(1): p. 56-71). However, the critical involvement of DNA methylation in shaping expansion, memory state status and thus functionality of antigen-specific TILs undergoing TCR ligation-dependent expansion ex vivo is not known (Abdelsamed, H.A., et al., J Exp Med, 2017. 214(6): p. 1593-1606).

[0287] There is evidence to show that pharmacological activation of the Wnt / (3- catenin signaling reprograms tumor-specific CD8+ TILs in tumor-bearing mice by increasing expression of Tcfl (encoded by Tcf7) (Gattinoni, L., et al., Nat Med, 2009. 15(7): p. 808-13).In human melanoma, CD8+TCF7+ uncovers a subset of TILs with a memory precursor-like cell state with bystander cytotoxic function and is associated with positive outcome to immune checkpoint blockade (Li, H., et al., Cell, 2019. 176(4): p. 775-789 el8; Sade-Feldman, M., et al., Cell, 2018. 175(4): p. 998-1013 e20). Down regulation of the components of the Wnt pathway has been reported in PD1+ CD8+ memory TILs in vivo and a further downregulation upon in vitro expansion in all T cell populations (Lipp, J.J., et al., Oncoimmunology, 2022. 11(1): p. 2019466), whereas compensating the lack of Wnt signaling through pharmacological inhibition of GSK30 improves the effector function of in vitro expanded populations. As Wnt signaling is known to arrest the development of effector CD8+ T cells, it also might be involved in epigenetically reprogramming TILs. GSK-3 is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function, and is effective in controlling tumor growth (Taylor, A., et al., Immunity, 2016. 44(2): p. 274-86; Steele, L., et al., iScience, 2021. 24(6): p. 102555). Besides being a central regulator of PD-1,GSK-3 also negatively regulates Lymphocyte Activation Gene-3 (LAG-3) expression on CD4+ and CD8+ T cells and small molecule inhibitors of GSK-3 were more effective than LAG-3 blockade alone in suppressing tumor growth in murine model of melanoma (Rudd, C.E., et al., Cell Rep, 2020. 30(7): p. 2075-2082 e4). More recently, it was demonstrated that Wnt activation promotes human memory T cell polyfunctionality via epigenetic regulator PRMT1 (Sung, B.Y., et al., J Clin Invest, 2022. 132(2)).

[0288] Therefore, in some embodiments, the second cell culture medium comprises an inhibitor of GSK-3a / p. In some embodiments, the inhibitor of GSK-3a / P is selected from SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8. In some embodiments, the inhibitor of GSK-3a / p is SB415286. In some embodiments, the inhibitor of GSK-3a / P is SB216763. In some embodiments, the inhibitor of GSK-3a / P is CHIR99021. In some embodiments, the inhibitor of GSK-3a / P is AR-AO14418. In some embodiments, the inhibitor of GSK-3a / P is TZD8. In some embodiments, the inhibitor of GSK-3a / P is TWS119. In some embodiments, the inhibitor of GSK-3a / P is Lithium Chloride hydrate. In some embodiments, the inhibitor of GSK-3a / P is BIO. In some embodiments, the inhibitor of GSK-3a / P is 3F8.H. Harvest TILs

[0289] After the second expansion step, cells can be harvested. 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.

[0290] Cell harvesters and / or cell processing systems are commercially available from a variety of 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, allowingfor 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.

[0291] In some embodiments, the harvest is performed from 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.

[0292] 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.I. Final Formulation and Transfer to Infusion Container

[0293] After the steps as outlined in detailed above and herein are complete, TILs 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, such as an infusion bag, for use in administration to a patient. In some embodiments, the TILs are cryopreserved in the infusion bag. In some embodiments, the TILs are cryopreserved prior to placement in an infusion bag. In some embodiments, the TILs are cryopreserved and not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation media contains dimethylsulfoxide (DMSO). This is generally accomplished by putting the TIL population into a freezing solution, e.g. 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed into cryogenic vials and stored for 24 hours at -80 °C, with optional transfer to gaseous nitrogen freezers for cryopreservation. See, Sadeghi, et al., Acta Oncologica 2013, 52, 978-986.

[0294] When appropriate, the cells are removed from the freezer and thawed in a 37 °C water bath until approximately 4 / 5 of the solution is thawed. The cells are generally resuspended in complete media and optionally washed one or more times. In someembodiments, the thawed TILs can be counted and assessed for viability as is known in the art.

[0295] In some embodiments, a population of TILs is cryopreserved using CS10 cryopreservation media (CryoStor 10, BioLife Solutions). In some embodiments, a population of TILs is cryopreserved using a cryopreservation media containing dimethylsulfoxide (DMSO). In some embodiments, a population of TILs is cryopreserved using a 1:1 (vokvol) ratio of CS10 and cell culture media. In some embodiments, a population of TILs is cryopreserved using about a 1:1 (vokvol) ratio of CS10 and cell culture media, further comprising additional IL-2.

[0296] In some embodiments, TILs 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 by methods described in 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.J. Closed Systems for TIL Manufacturing

[0297] Closed systems may be used during the TIL culturing process. Such closed systems allow for preventing and / or reducing microbial contamination, allow for the use of fewer flasks, and allow for cost reductions. In some embodiments, the closed system uses two containers. Such closed systems are well-known in the art and can be found, for example, at fda.gov / cber / guidelines.htm and fda.gov / BiologicsBlood Vaccines / GuidanceComplianceRegulatorylnformation / Guidances / Blo od / ucm076779.htm.

[0298] Sterile connecting devices (STCDs) produce sterile welds between two pieces of compatible tubing. This procedure permits sterile connection of a variety of containers and tube diameters. In some embodiments, the closed systems include luer lock and heat-sealed systems as described in the Examples. 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 isemployed. In some embodiments, the TILs are formulated into a final product formulation container according to the methods described herein in the examples.

[0299] In some embodiments, the closed system uses one container from the time the tumor fragments are obtained until the TILs are ready for administration to the patient or cryopreserving. In some embodiments when two containers are used, the first container is a closed G-container (such as G-rex10OM series or G-rex500M series flasks) and the population of TILs is centrifuged and transferred to an infusion bag without opening the first closed G-container. In some embodiments, when two containers are used, the infusion bag is a HypoThermosol-containing infusion bag. A closed system or closed TIL cell culture system is characterized in that once the tumor sample and / or tumor fragments have been added, the system is tightly sealed from the outside to form a closed environment free from the invasion of bacteria, fungi, and / or any other microbial contamination.

[0300] In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

[0301] The closed system allows for TIL growth in the absence and / or with a significant reduction in microbial contamination.

[0302] Moreover, pH, carbon dioxide partial pressure and oxygen partial pressure of the TIL cell culture environment each vary as the cells are cultured. Consequently, even though a medium appropriate for cell culture is circulated, the closed environment still needs to be constantly maintained as an optimal environment forTIL proliferation. To this end, it is desirable that the physical factors of pH, carbon dioxide partial pressure and oxygen partial pressure within the culture liquid of the closed environment be monitored by means of asensor, the signal whereof is used to control a gas exchanger installed at the inlet of the culture environment, and the that gas partial pressure of the closed environment be adjusted in real time according to changes in the culture liquid so as to optimize the cell culture environment. In some embodiments, a closed cell culture system is used which incorporates at the inlet to the closed environment a gas exchanger equipped with a monitoring device which measures the pH, carbon dioxide partial pressure and oxygen partial pressure of the closed environment, and optimizes the cell culture environment by automatically adjusting gas concentrations based on signals from the monitoring device.

[0303] In some embodiments, the pressure within the closed environment is continuously or intermittently controlled. That is, the pressure in the closed environment can be varied by means of a pressure maintenance device for example, thus ensuring that the space is suitable for growth of TILs in a positive pressure state, or promoting exudation of fluid in a negative pressure state and thus promoting cell proliferation. By applying negative pressure intermittently, moreover, it is possible to uniformly and efficiently replace the circulating liquid in the closed environment by means of a temporary shrinkage in the volume of the closed environment.

[0304] In some embodiments, additional equipment, such as an electroporator (e.g., a Neon electroporator) is a component of an all-closed system. In some embodiments, optimal culture components for proliferation of the TILs can be substituted or added, and including factors such as IL-2 and / or OKT3, as well as combination, can be added.

[0305] In other embodiments, provided herein is the method described in any of the preceding paragraphs as applicable above modified such that each container recited in the method is a GREX-10. In other embodiments, the method described in any of the preceding paragraphs as applicable above is modified such that each container recited in the method is a GREX-100M. In other embodiments, the method described in any of the preceding paragraphs as applicable above is modified such that each container recited in the method is a GREX-500M.IV. Pharmaceutical Compositions, Dosages, and Dosing Regimens

[0306] In some embodiments, TILs expanded using the methods of the present disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, thepharmaceutical 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 intra lymphatic administration.

[0307] Accordingly, some embodiments of the present disclosure provide a therapeutic population of TILs manufactured using the method disclosed herein. In some embodiments, the therapeutic population of TILs comprises from about 1x109to about 1x1011TILs. In some embodiments, the therapeutic population of TILs exhibits enhanced polyfunctionality in comparison to a population of TILs manufactured using a reference TIL manufacturing process. In some embodiments, the therapeutic population of TILs exhibits a more stem-like phenotype in comparison to a population of TILs manufactured using a reference TIL manufacturing process. In some embodiments, the therapeutic population of TILs exhibits an increase in the frequency of less activated and / or differentiated TILs in comparison to a population of TILs manufactured using a reference TIL manufacturing process. In some embodiments, the therapeutic population of TILs exhibits improved tumor cell killing in an allogeneic setting in comparison to a population of TILs manufactured using a reference TIL manufacturing process. In some embodiments, the therapeutic population of TILs exhibits increased expression of a memory associated marker selected from CD27, CD28, CD62L, and IL-7R, in comparison to a population of TILs manufactured using a reference TIL manufacturing process. In some embodiments, the therapeutic population of TILs exhibits reduced expression of an activation marker selected from CD38, CD39, and CD69, in comparison to a population of TILs manufactured using a reference TIL manufacturing process. In some embodiments, the therapeutic population of TILs exhibits reduced expression of an inhibitory / exhaustion associated marker selected from LAG3, TIM3, TIGIT, and TOX, in comparison to a population of TILs manufactured using a reference TIL manufacturing process. In some embodiments, the therapeutic population of TILs exhibits increased expression of a functional marker selected from GZMB, CXCR3, 1 FNg, TNFa, and IL- 2, in comparison to a population of TILs manufactured using a reference TIL manufacturingprocess. In some embodiments, the reference TIL manufacturing process is a TIL manufacturing process disclosed herein, such as a Gen 2 process or process 2A.

[0308] In some embodiments, the number of the TILs provided in the pharmaceutical compositions is about 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, 5x1010, 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013, and 9x1013. In some embodiments, the number of the TILs provided in the pharmaceutical compositions is in the range of 1x106to 5x106, 5x106to 1x107, 1x107to 5x107, 5x107to 1x108, 1x108to 5x108, 5x108to 1x109, 1x109to 5x109, 5x109to 1x1010, 1x1010to 5x1010, 5x1010to 1x1011, 5x1011to 1x1012, 1x1012to 5x1012, and 5x1012to 1x1013.

[0309] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v of the pharmaceutical composition.

[0310] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%,16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%,13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%,10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%,6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v, or v / v of the pharmaceutical composition.

[0311] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w / w, w / v or v / v of the pharmaceutical composition.

[0312] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0313] In some embodiments, the amount of the TILs provided in the pharmaceutical compositions is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g,5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

[0314] In some embodiments, the amount of the TILs provided in the pharmaceutical compositions is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g,1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

[0315] The TILs provided in the pharmaceutical compositions are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.

[0316] In some embodiments, TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as long as necessary.

[0317] In some embodiments, an effective dosage of TILs is about 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, 5x1010, 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013, and 9x1013. In some embodiments, an effective dosage of TILs is in the range of 1x106to 5x106, 5x106to 1x107, 1x107to 5x107, 5x107to 1x108, 1x108to 5x108, 5x108to 1x109, 1x109to 5x109, 5x109to 1x1010, 1x1010to 5x1010, 5x1010to 1x1011, 5x1011to 1x1012, 1x1012to 5x1012, and 5x1012to 1x1013.

[0318] In some embodiments, an effective dosage of TILs is in the range of about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg,about 0.3 mg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg mg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0319] In some embodiments, an effective dosage of TILs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

[0320] An effective amount of the TILs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation.

[0321] In other embodiments, provided herein is an infusion bag comprising the therapeutic population of TILs described in any of the preceding paragraphs above.

[0322] In other embodiments, provided herein is a tumor infiltrating lymphocyte (TIL) composition comprising the therapeutic population of TILs described in any of the preceding paragraphs above and a pharmaceutically acceptable carrier.

[0323] In other embodiments, provided herein is an infusion bag comprising the TIL composition described in any of the preceding paragraphs above.

[0324] In other embodiments, provided herein is a cryopreserved preparation of the therapeutic population of TILs described in any of the preceding paragraphs above.

[0325] In other embodiments, provided herein is a tumor infiltrating lymphocyte (TIL) composition comprising the therapeutic population of TILs described in any of the preceding paragraphs above and a cryopreservation media.

[0326] In other embodiments, provided herein is the TIL composition described in any of the preceding paragraphs above modified such that the cryopreservation media contains DMSO.

[0327] In other embodiments, provided herein is the TIL composition described in any of the preceding paragraphs above modified such that the cryopreservation media contains 7-10% DMSO.

[0328] In other embodiments, provided herein is a cryopreserved preparation of the TIL composition described in any of the preceding paragraphs above.

[0329] In some embodiments, TILs expanded using the methods of the present disclosure are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of 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.

[0330] Any suitable dose of TILs can be administered. In some embodiments, from about 2.3x1010to about 13.7x10loTILs are administered, with an average of around 7.8x10loTILs, particularly if the cancer is NSCLC. In some embodiments, about 1.2x1010to about 4.3x1010of TILs are administered. In some embodiments, about 3x1010to about 12x1010TILs are administered. In some embodiments, about 4x1010to about 10x10loTILs are administered. In some embodiments, about 5x1010to about 8x10loTILs are administered. In some embodiments, about 6x1010to about 8x10loTILs are administered. In some embodiments, about 7x1010to about 8x10loTILs are administered. In some embodiments, therapeutically effective dosage is about 2.3x1010to about 13.7x1010. In some embodiments,therapeutically effective dosage is about 7.8x10loTILs, particularly of the cancer is NSCLC. In some embodiments, therapeutically effective dosage is about 1.2x1010to about 4.3x1010ofTILs. In some embodiments, therapeutically effective dosage is about 3x1010to about 12x10loTILs. In some embodiments, therapeutically effective dosage is about 4x1010to about 10x1010TILs. In some embodiments, therapeutically effective dosage is about 5x1010to about 8x10loTILs. In some embodiments, therapeutically effective dosage is about 6x1010to about 8x10loTILs. In some embodiments, therapeutically effective dosage is about 7x1010to about 8x10loTILs.

[0331] In some embodiments, the number of the TILs provided in the pharmaceutical compositions is about 1x106, 2x106, 3x106, 4x105, 5x106, 6x106, 7x105, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, 5x1010, 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013, and 9x1013. In some embodiments, the number of the TILs provided in the pharmaceutical compositions is in the range of 1x106to 5x106, 5x10sto 1x107, 1x107to 5x107, 5x107to 1x108, 1x108to 5x108, 5x108to 1x109, 1x109to 5x109, 5x109to 1x1010, 1x1010to 5x1010, 5x1010to 1x1011, 5x1011to 1x1012, 1x1012to 5x1012, and 5x1012to 1x1013.

[0332] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v of the pharmaceutical composition.

[0333] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%,13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%,10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v, or v / v of the pharmaceutical composition.

[0334] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w / w, w / v or v / v of the pharmaceutical composition.

[0335] In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0336] In some embodiments, the amount of the TILs provided in the pharmaceutical compositions is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

[0337] In some embodiments, the amount of the TILs provided in the pharmaceutical compositions is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

[0338] The TILs provided in the pharmaceutical compositions are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.

[0339] In some embodiments, TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as long as necessary.

[0340] In some embodiments, an effective dosage of TILs is about 1x105, 2x106, 3x105, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1x10s, 2x10s, 3x10s, 4x10s, 5x10s, 6x10s, 7x10s, 8x10s, 9x10s, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, 5x1010, 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8X1011, 9X1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012,1x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013, and 9x1013. In some embodiments, an effective dosage of TILs is in the range of 1x106to 5x106, 5x106to 1x107, 1x107to 5X107, 5x107to 1X108, 1x108to 5x108, 5x108to 1x109, 1x109to 5x109, 5x109to 1X1O10, 1X1O10to 5x1010, 5x1010to 1x1011, 5x1011to 1x1012, 1x1012to 5x1012, and 5x1012to 1x1013.

[0341] In some embodiments, an effective dosage of TILs is in the range of about 0.01 mg / kg to about 4.3 mg / kg, about 0.15 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about3.2 mg / kg, about 0.35 mg / kg to about 2.85 mg / kg, about 0.15 mg / kg to about 2.85 mg / kg, about 0.3 mg to about 2.15 mg / kg, about 0.45 mg / kg to about 1.7 mg / kg, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg mg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0342] In some embodiments, an effective dosage of TILs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

[0343] An effective amount of the TILs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously,intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation.V. Methods of Treating Patients

[0344] Methods of treatment begin with the initial TIL collection and culture of TILs. Such methods have been both described in the art by, for example, Jin et al., J. Immunotherapy, 2012, 35(3):283-292, incorporated by reference herein in its entirety. Embodiments of methods of treatment are described throughout the sections below, including the Examples.

[0345] The expanded TILs produced according the methods described herein, find particular use in the treatment of patients with cancer (for example, as described in Goff, et al., J. Clinical Oncology, 2016, 34(20):2389-239, as well as the supplemental content; incorporated by reference herein in its entirety. In some embodiments, TIL were grown from resected deposits of metastatic melanoma as previously described (see, Dudley, et al., J Immunother., 2003, 26:332-342; incorporated by reference herein in its entirety). Fresh tumor can be dissected under sterile conditions. A representative sample can be collected for formal pathologic analysis. Single fragments of 2 mm3to 3 mm3may be used. In some embodiments, 5, 10, 15, 20, 25 or 30 samples per patient are obtained. In some embodiments, 20, 25, or 30 samples per patient are obtained. In some embodiments, 20, 22, 24, 26, or 28 samples per patient are obtained. In some embodiments, 24 samples per patient are obtained. Samples can be placed in individual wells of a 24-well plate, maintained in growth media with high-dose IL-2 (6,000 lU / mL), and monitored for destruction of tumor and / or proliferation of TIL. Any tumor with viable cells remaining after processing can be enzymatically digested into a single cell suspension and cryopreserved, as described herein.

[0346] In some embodiments, successfully grown TIL can be sampled for phenotype analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumor when available. TIL can be considered reactive if overnight coculture yielded interferon-gamma (IFN-y) levels > 200 pg / mL and twice background. (Goff, et al., J Immunother., 2010, 33:840-847; incorporated by reference herein in its entirety). In some embodiments, cultures with evidence of autologous reactivity or sufficient growth patterns can be selected for a second expansion, including second expansions that are sometimes referred to as rapid expansion (REP). In someembodiments, expanded TILs with high autologous reactivity (for example, high proliferation during a second expansion), are selected for an additional second expansion. In some embodiments, TILs with high autologous reactivity, are selected for an additional second expansion.

[0347] Cell phenotypes of cryopreserved samples of infusion bag TIL can be analyzed by flow cytometry (e.g., FlowJo) for surface markers CDS, CD4, CD8, CCR7, and CD45RA (BD BioSciences), as well as by any of the methods described herein. Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. A rise in serum IFN-g was defined as >100 pg / mL and greater than 4 3 baseline levels.

[0348] Measures of efficacy can include the disease control rate (DCR) as well as overall response rate (ORR), as known in the art as well as described herein.A. Methods of Treating Cancers

[0349] The compositions and methods described herein can be used in a method for treating diseases. In some embodiments, they are for use in treating hyperproliferative disorders, such as cancer, in an adult patient or in a pediatric patient. They may also be used in treating other disorders as described herein and in the following paragraphs.

[0350] In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papilloma virus (HPV), central nervous system 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 uveal melanoma, choroidal melanoma, ciliary body melanoma, or irismelanoma), 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.

[0351] In some embodiments, the hyperproliferative disorder is a hematological malignancy. In some embodiments, the hematological malignancy is chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the cancer is a hematological malignancy. In some embodiments, provided herein is a method of treating a patient with a cancer using TILs, MILs, or PBLs modified to express one or more CCRs, wherein the cancer is a hematological malignancy. In some embodiments, provided herein is a method of treating a patient with a cancer using MILs or PBLs modified to express one or more CCRs, wherein the cancer is a hematological malignancy.

[0352] In some embodiments, the cancer is one of the foregoing cancers, including solid tumor cancers and hematological malignancies, that is relapsed or refractory to treatment with at least one prior therapy, including chemotherapy, radiation therapy, or immunotherapy. In some embodiments, the cancer is one of the foregoing cancers that is relapsed or refractory to treatment with at least two prior therapies, including chemotherapy, radiation therapy, and / or immunotherapy. In some embodiments, the cancer is one of the foregoing cancers that is relapsed or refractory to treatment with at least three prior therapies, including chemotherapy, radiation therapy, and / or immunotherapy.

[0353] In some embodiments, the cancer is a microsatellite instability-high (MSI-H) or a mismatch repair deficient (dMMR) cancer. MSI-H and dMMR cancers and testing therefore have been described in Kawakami, et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosures of which are incorporated by reference herein.

[0354] In some embodiments, provided herein is a method of treating a patient with a cancer using TILs, MILs, or PBLs modified to express one or more CCRs, wherein the patientis a human. In some embodiments, provided herein is a method of treating a patient with a cancer using TILs, MILs, or PBLs modified to express one or more CCRs, wherein the patient is a non-human. In some embodiments, provided herein is a method of treating a patient with a cancer using TILs, MILs, or PBLs modified to express one or more CCRs, wherein the patient is a companion animal.

[0355] In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor and / or a MEK inhibitor. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor selected from vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the cancer is refractory to treatment with a MEK inhibitor selected from trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor selected from vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof, and a MEK inhibitor selected from trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof.

[0356] In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the cancer is a pediatric cancer. In some embodiments, provided herein is a method of treating a patient with a cancer wherein the cancer is uveal melanoma. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the uveal melanoma is choroidal melanoma, ciliary body melanoma, or iris melanoma. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the pediatric cancer is a neuroblastoma.

[0357] In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the cancer is a sarcoma. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the sarcoma is osteosarcoma. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein thesarcoma is a soft tissue sarcoma. In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing sarcoma, or primitive neuroectodermal tumor (PNET).

[0358] In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the pediatric cancer is a central nervous system (CNS) associated cancer. In some embodiments, the pediatric cancer is refractory to treatment with chemotherapy. In some embodiments, the pediatric cancer is refractory to treatment with radiation therapy. In some embodiments, the pediatric cancer is refractory to treatment with dinutuximab.

[0359] In some embodiments, provided herein is a method of treating a patient with a cancer, wherein the CNS associated cancer is medulloblastoma, pineoblastoma, glioma, ependymoma, or glioblastoma.

[0360] The compositions and methods described herein can be used in a method for treating cancer, wherein the cancer is refractory or resistant to prior treatment with an anti- PD-1 or anti-PD-Ll antibody. In some embodiments, the patient is a primary refractory patient to an anti-PD-1 or anti-PD-Ll antibody. In some embodiments, the patient shows no prior response to an anti-PD-1 or anti-PD-Ll antibody. In some embodiments, the patient shows a prior response to an anti-PD-1 or anti-PD-Ll antibody, follow by progression of the patient's cancer. In some embodiments, the cancer is refractory to an anti-CTLA-4 antibody and / or an anti-PD-1 or anti-PD-Ll antibody in combination with at least one chemotherapeutic agent. In some embodiments, the prior chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and / or cisplatin. In some prior embodiments, the chemotherapeutic agent(s) is a platinum doublet chemotherapeutic agent. In some embodiments, the platinum doublet therapy comprises a first chemotherapeutic agent selected from cisplatin and carboplatin and a second chemotherapeutic agent selected from vinorelbine, gemcitabine and a taxane (including for example, paclitaxel, docetaxel or nab- paclitaxel). In some embodiments, the platinum doublet chemotherapeutic agent is in combination with pemetrexed.

[0361] In some embodiments, the NSCLC is PD-L1 negative and / or is from a patient with a cancer that expresses PD-L1 with a tumor proportion score (TPS) of < 1%, as described elsewhere herein.

[0362] In some embodiments, the NSCLC is refractory to a combination therapy comprising an anti-PD-1 or the anti-PD-Ll antibody and a platinum doublet therapy, wherein the platinum doublet therapy comprises: i) a first chemotherapeutic agent selected from cisplatin and carboplatin, ii) and a second chemotherapeutic agent selected from vinorelbine, gemcitabine and a taxane (including for example, paclitaxel, docetaxel or nab-paclitaxel).

[0363] In some embodiments, the NSCLC is refractory to a combination therapy comprising an anti-PD-1 or the anti-PD-Ll antibody, pemetrexed, and a platinum doublet therapy, wherein the platinum doublet therapy comprises: iii) a first chemotherapeutic agent selected from cisplatin and carboplatin, iv) and a second chemotherapeutic agent selected from vinorelbine, gemcitabine and a taxane (including for example, paclitaxel, docetaxel or nab-paclitaxel).

[0364] In some embodiments, the NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has been treated with an anti-PD-Ll antibody. In some embodiments, the NSCLC patient is treatment naive. In some embodiments, the NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has not been treated with an anti-PD-Ll antibody. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent but is not longer being treated with the chemotherapeutic agent. In some embodiments, the NSCLC patient is anti-PD-l / PD-Ll naive. In some embodiments, the NSCLC patient has low expression of PD-L1. In some embodiments, the NSCLC patient has treatment naive NSCLC or is post-chemotherapeutic treatment but anti-PD-l / PD-Ll naive. In some embodiments, the NSCLC patient is treatment naive or post-chemotherapeutic treatment but anti-PD-l / PD-Ll naive and has low expression of PD-L1. In some embodiments, the NSCLC patient has bulky disease at baseline. In some embodiments, the subject has bulky disease at baseline and has low expression of PD-L1. In some embodiments, the NSCLC patient has no detectable expression of PD-L1. In some embodiments, the NSCLC patient is treatment naive or post-chemotherapeutic treatment but anti-PD-l / PD-Ll naive and has no detectable expression of PD-L1. In someembodiments, the patient has bulky disease at baseline and has no detectable expression ofPD-L1. In some embodiments, the NSCLC patient has treatment naive NSCLC or post chemotherapy (e.g., post chemotherapeutic agent) but anti-PD-l / PD-Ll naive who have low expression of PD-L1 and / or have bulky disease at baseline. In some embodiments, bulky disease is indicated where the maximal tumor diameter is greater than 7 cm measured in either the transverse or coronal plane. In some embodiments, bulky disease is indicated when there are swollen lymph nodes with a short-axis diameter of 20 mm or greater. In some embodiments, the chemotherapeutic includes a standard of care therapeutic for NSCLC.

[0365] In some embodiments, PD-L1 expression is determined by the tumor proportion score. In some embodiments, the subject with a refractory NSCLC tumor has a < 1% tumor proportion score (TPS). In some embodiments, the subject with a refractory NSCLC tumor has a > 1% TPS. In some embodiments, subject with the refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-Ll antibody and the tumor proportion score was determined prior to said anti-PD-1 and / or anti-PD-Ll antibody treatment. In some embodiments, subject with the refractory NSCLC has been previously treated with an anti- PD-Ll antibody and the tumor proportion score was determined prior to said anti-PD-Ll antibody treatment.

[0366] In some embodiments, PD-L1 expression is determined by the tumor proportion score using one more testing methods as described herein. In some embodiments, the subject or patient with a NSCLC tumor has a < 1% tumor proportion score (TPS). In some embodiments, the NSCLC tumor has a > 1% TPS. In some embodiments, the subject or patient with the NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-Ll antibody and the tumor proportion score was determined prior to the anti-PD-1 and / or anti- PD-Ll antibody treatment. In some embodiments, the subject or patient with the NSCLC has been previously treated with an anti-PD-Ll antibody and the tumor proportion score was determined prior to the anti-PD-Ll antibody treatment. In some embodiments, the subject or patient with a refractory or resistant NSCLC tumor has a < 1% tumor proportion score (TPS). In some embodiments, the subject or patient with a refractory or resistant NSCLC tumor has a > 1% TPS. In some embodiments, the subject or patient with the refractory or resistant NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-Ll antibodyand the tumor proportion score was determined prior to the anti-PD-1 and / or anti-PD-Ll antibody treatment. In some embodiments, the subject or patient with the refractory or resistant NSCLC has been previously treated with an anti-PD-Ll antibody and the tumor proportion score was determined prior to the anti-PD-Ll antibody treatment.

[0367] In some embodiments, the NSCLC is an NSCLC that exhibits a tumor proportion score (TPS), or the percentage of viable tumor cells from a patient taken prior to anti-PD-1 or anti- PD-Ll therapy, showing partial or complete membrane staining at any intensity, for the PD- L1 protein that is less than 1% (TPS < 1%). In some embodiments, the NSCLC is an NSCLC that exhibits a TPS selected from <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%,<9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, <1%, <0.9%, <0.8%, <0.7%, <0.6%, <0.5%, <0.4%,<0.3%, <0.2%, <0.1%, <0.09%, <0.08%, <0.07%, <0.06%, <0.05%, <0.04%, <0.03%, <0.02%, and <0.01%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS selected from about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, and about 0.01%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 1%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.9%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.8%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.7%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.6%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.5%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.4%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.3%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.2%. In some embodiments, the NSCLC is an NSCLC that exhibits a TPS between 0% and 0.1%. TPS may be measured by methods known in the art, such as those described in Hirsch, et al. J. Thorac.Oncol. 2017, 12, 208-222 or those used for the determination of TPS prior to treatment with pembrolizumab or other anti-PD-1 or anti-PD-Ll therapies. Methods for measurement of TPS that have been approved by the U.S. Food and Drug Administration may also be used. Insome embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, the PD-L1 is found on circulating tumor cells.

[0368] In some embodiments, the partial membrane staining includes 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more. In some embodiments, the completed membrane staining includes approximately 100% membrane staining.

[0369] In some embodiments, testing for PD-L1 can involve measuring levels of PD-L1 in patient serum. In these embodiments, measurement of PD-L1 in patient serum removes the uncertainty of tumor heterogeneity and the patient discomfort of serial biopsies.

[0370] In some embodiments, elevated soluble PD-L1 as compared to a baseline or standard level correlates with worsened prognosis in NSCLC. See, for example, Okuma, et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli, et al., Oncotarget, 2018, 9, 17554-17563. In some embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, the PD-L1 is expressed on circulating tumor cells.

[0371] In some embodiments, provided herein is a method of treating cancer, e.g., nonsmall cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a subject or patient in need thereof, wherein the subject or patient has at least one of: a predetermined tumor proportion score (TPS) of PD-L1 < 1%, a TPS score of PD-L1 of l%-49%, or a predetermined absence of one or more driver mutations, wherein the driver mutation is selected from an EGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c- ROS mutation (R0S1 mutation), a R0S1 fusion, a RET mutation, a RET fusion, an ERBB2 mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation, PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, a KRAS mutation, an NF1 mutation, a MET mutation, a MET splice and / or altered MET signaling, a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D mutation, an ARID1A mutation, a RBI mutation, an ATM mutation, a SETD2mutation, a FLT3 mutation, a PTPN11 mutation, a FGFR1 mutation, an EP300 mutation, a MYC mutation, an EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, a CCND3 mutation, and a GNA11 mutation, and wherein the method comprises:(a) obtaining and / or receiving a first population of TILs from a tumor resected from the subject or patient by processing a tumor sample obtained from the subject into multiple tumor fragments;(b) adding the first population of TILs into a closed system;(c) 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 in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system;(d) 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 is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system;(e) harvesting therapeutic population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and(f) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system;(g) cryopreserving the infusion bag comprising the harvested TIL population from step (f) using a cryopreservation process; and(h) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject or patient.

[0372] In some embodiments, provided herein is a method of treating cancer, e.g., nonsmall cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, wherein the method comprises:(a) testing the patient's tumor for PD-L1 expression and tumor proportion score (TPS) of PD-L1,(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from an EGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion, a RET mutation, a RET fusion, an ERBB2 mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation, PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, a KRAS mutation, an NF1 mutation, a MET mutation, a MET splice and / or altered MET signaling, a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D mutation, an ARID1A mutation, a RBI mutation, an ATM mutation, a SETD2 mutation, a FLT3 mutation, a PTPN11 mutation, a FGFR1 mutation, an EP300 mutation, a MYC mutation, an EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, a CCND3 mutation, and a GNA11 mutation,(c) determining that the patient has a TPS score for PD-L1 of about 1% to about 49% and determining that the patient also has no driver mutations,(d) obtaining and / or receiving a first population of TILs from a tumor resected from the subject or patient by processing a tumor sample obtained from the subject into multiple tumor fragments;(e) adding the first population of TILs into a closed system;(f) 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 in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) occurs without opening the system;(g) 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 is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system;(h) harvesting therapeutic population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and(i) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system;(j) cryopreserving the infusion bag comprising the harvested TIL population from step (f) using a cryopreservation process; and(k) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject or patient.

[0373] In some embodiments, provided herein is a method of treating cancer, e.g., nonsmall cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, wherein the method comprises:(a) testing the patient's tumor for PD-L1 expression and tumor proportion score (TPS) of PD-L1,(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from an EGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion, a RET mutation, a RET fusion, an ERBB2 mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation, PIK3CA, CDKN2A, a PTEN mutation, an UMD mutation, an NRAS mutation, a KRAS mutation, an NF1 mutation, a MET mutation, a MET splice and / or altered MET signaling, a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D mutation, an ARID1Amutation, a RBI mutation, an ATM mutation, a SETD2 mutation, a FLT3 mutation, a PTPN11 mutation, a FGFR1 mutation, an EP3OO mutation, a MYC mutation, an EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, a CCND3 mutation, and a GNA11 mutation,(c) determining that the patient has a TPS score for PD-L1 of less than about 1% and determining that the patient also has no driver mutations,(d) obtaining and / or receiving a first population of TILs from a tumor resected from the subject or patient by processing a tumor sample obtained from the subject into multiple tumor fragments;(e) adding the first population of TILs into a closed system;(f) 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 in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) occurs without opening the system;(g) 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 is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system;(h) harvesting therapeutic population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and(i) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system;(j) cryopreserving the infusion bag comprising the harvested TIL population from step (f) using a cryopreservation process; and(k) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject or patient.

[0374] In some embodiments, provided herein is a method of treating cancer, e.g., nonsmall cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, wherein the method comprises:(a) testing the patient's tumor for PD-L1 expression and tumor proportion score (TPS) of PD-L1,(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from an EGFR mutation, an EGFR insertion, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion, a RET mutation, or a RET fusion,(c) determining that the patient has a TPS score for PD-L1 of about 1% to about 49% and determining that the patient also has no driver mutations,(d) obtaining and / or receiving a first population of TILs from a tumor resected from the subject or patient by processing a tumor sample obtained from the subject into multiple tumor fragments;(e) adding the first population of TILs into a closed system;(f) 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 in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) occurs without opening the system;(g) 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 isperformed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system;(h) harvesting therapeutic population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and(i) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system;(j) cryopreserving the infusion bag comprising the harvested TIL population from step (f) using a cryopreservation process; and(k) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject or patient.

[0375] In some embodiments, provided herein is a method of treating cancer, e.g., nonsmall cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, wherein the method comprises:(a) testing the patient's tumor for PD-L1 expression and tumor proportion score (TPS) of PD-L1,(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from an EGFR mutation, an EGFR insertion, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion, a RET mutation, or a RET fusion,(c) determining that the patient has a TPS score for PD-L1 of less than about 1% and determining that the patient also has no driver mutations,(d) obtaining and / or receiving a first population of TILs from a tumor resected from the subject or patient by processing a tumor sample obtained from the subject into multiple tumor fragments;(e) adding the first population of TILs into a closed system;(f) 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 in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, and wherein the transition from step (e) to step (f) occurs without opening the system;(g) 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 is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system;(h) harvesting therapeutic population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and(i) transferring the harvested TIL population from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system;(j) cryopreserving the infusion bag comprising the harvested TIL population from step (f) using a cryopreservation process; and(k) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject or patient.

[0376] In other embodiments, provided herein is a method for treating a subject with cancer comprising administering to the subject a therapeutically effective dosage of the therapeutic TIL population described herein. In other embodiments, provided herein is a method for treating a subject with cancer comprising administering to the subject a therapeutically effective dosage of the TIL composition described herein.

[0377] In other embodiments, the method for treating a subject with cancer described herein is modified such that prior to administering the therapeutically effective dosage ofthe therapeutic TIL population and the TIL composition described herein, respectively, a non-myeloablative lymphodepletion regimen has been administered to the subject. In other embodiments, the method for treating a subject with cancer described herein is modified such that 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.

[0378] In other embodiments, the method for treating a subject with cancer described herein is modified to further comprise the step of treating the subject with a high-dose IL-2 regimen starting on the day after administration of the TIL cells to the subject. In other embodiments, the method for treating a subject with cancer described herein is modified such that the high-dose IL-2 regimen comprises 600,000 or 720,000 lU / kg administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.

[0379] In other embodiments, the cancer is a solid tumor. In other embodiments, the cancer is melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

[0380] In other embodiments, the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer. In other embodiments, the cancer is melanoma. In other embodiments, the cancer is HNSCC. In other embodiments, the cancer is a cervical cancer. In other embodiments, the cancer is NSCLC. In other embodiments, the cancer is glioblastoma (including GBM). In other embodiments, the cancer is gastrointestinal cancer. In other embodiments, the cancer is a hypermutated cancer. In other embodiments, the cancer is a pediatric hypermutated cancer.

[0381] In other embodiments, a therapeutic TIL population described herein is for use in a method for treating a subject with cancer comprising administering to the subject a therapeutically effective dosage of the therapeutic TIL population.

[0382] In other embodiments, a TIL composition described herein is for use in a method for treating a subject with cancer comprising administering to the subject a therapeutically effective dosage of the TIL composition.

[0383] In other embodiments, prior to administering to the subject the therapeutically effective dosage of the therapeutic TIL population described herein or the TIL composition described herein, a non-myeloablative lymphodepletion regimen has been administered to the subject. In other 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.

[0384] In other embodiments, the patient is treated with a high-dose IL-2 regimen starting on the day after administration of the TIL cells to the patient. In other embodiments, the high-dose IL-2 regimen comprises 600,000 or 720,000 lU / kg administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.

[0385] In other embodiments, a therapeutically effective dosage of the therapeutic TIL population is administered to the subject. In other embodiments, a therapeutically effective dosage of the TIL composition is administered to the subject.

[0386] In other embodiments, provided herein is the use of a therapeutic TIL population described herein or a TIL composition described herein in a method of treating cancer in a patient comprising administering to the patient a non-myeloablative lymphodepletion regimen and then administering to the subject the therapeutically effective dosage of the therapeutic TIL population described in any of the preceding paragraphs or the therapeutically effective dosage of the TIL composition described herein.A. Lymphodepletion Preconditioning of Patients

[0387] In some embodiments, provided herein is a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure. In some embodiments, provided herein is a population of TILs for use in the treatment of cancer in a patient which has been pre-treated with non-myeloablative chemotherapy. In some embodiments, the population of TILs is for administration by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days T1 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) according to the present disclosure, the patient receives an intravenous infusion of IL- 2 (aldesleukin, commercially available as PROLEUKIN) intravenously at 720,000 lU / kg every 8 hours to physiologic tolerance. In certain embodiments, the population of TILs is for use in treating cancer in combination with IL-2, wherein the IL-2 is administered after the population of TILs.

[0388] 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, in some embodiments a lymphodepletion step (sometimes also referred to as "immunosuppressive conditioning") is utilized on the patient prior to the introduction of the TILs.

[0389] In general, lymphodepletion is achieved using administration of fludarabine or cyclophosphamide (the active form being referred to as mafosfamide) and combinations thereof. Such methods are described in Gassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated by reference herein in their entireties.

[0390] In some embodiments, the fludarabine is administered at a concentration of 0.5 pg / mL to 10 pg / mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of 1 pg / mL fludarabine. In some embodiments, the fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the fludarabine is administered at a dosage of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / dayJ25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day. In some embodiments, the fludarabine treatment is administered for 2- 7 days at 35 mg / kg / day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 35 mg / kg / day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 25 mg / kg / day.

[0391] In some embodiments, the mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 0.5 pg / mL to 10 pg / mL by administration of cyclophosphamide. In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 1 pg / mL by administration of cyclophosphamide. In some embodiments, the cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the cyclophosphamide is administered at a dosage of 100 mg / m2 / day, 150 mg / m2 / day, 175 mg / m2 / dayj200 mg / m2 / day, 225 mg / m2 / day, 250 mg / m2 / day, 275 mg / m2 / day, or 300 mg / m2 / day. In some embodiments, the cyclophosphamide is administered intravenously (i.e., i.v.) In some embodiments, the cyclophosphamide treatment is administered for 2-7 days at 35 mg / kg / day. In some embodiments, the cyclophosphamide treatment is administered for 4-5 days at 250 mg / m2 / day i.v. In some embodiments, the cyclophosphamide treatment is administered for 4 days at 250 mg / m2 / day i.v.

[0392] In some embodiments, lymphodepletion is performed by administering the fludarabine and the cyclophosphamide together to a patient. In some embodiments, fludarabine is administered at 25 mg / m2 / day i.v. and cyclophosphamide is administered at 250 mg / m2 / day i.v. over 4 days.

[0393] In some embodiments, the lymphodepletion is performed by 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.

[0394] In some embodiments, the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days and administration of fludarabine at a dose of 25 mg / m2 / day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein the lymphodepletion is performed in five days in total.

[0395] In some embodiments, the lymphodepletion is performed by administration of cyclophosphamide at a dose of about 50 mg / m2 / day for two days and administration of fludarabine at a dose of about 25 mg / m2 / day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein the lymphodepletion is performed in five days in total.

[0396] In some embodiments, the lymphodepletion is performed by administration of cyclophosphamide at a dose of about 50 mg / m2 / day for two days and administration of fludarabine at a dose of about 20 mg / m2 / day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein the lymphodepletion is performed in five days in total.

[0397] In some embodiments, the lymphodepletion is performed by administration of cyclophosphamide at a dose of about 40 mg / m2 / day for two days and administration of fludarabine at a dose of about 20 mg / m2 / day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein the lymphodepletion is performed in five days in total.

[0398] In some embodiments, the lymphodepletion is performed by administration of cyclophosphamide at a dose of about 40 mg / m2 / day for two days and administration of fludarabine at a dose of about 15 mg / m2 / day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein the lymphodepletion is performed in three days in total.

[0399] In some embodiments, the lymphodepletion is performed by 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.

[0400] In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, mesna is administered at 15 mg / kg. In some embodiments where mesna is infused, and if infused continuously, mesna can be infused over approximately 2 hours with cyclophosphamide (on Days -5 and / or -4), then at a rate of 3 mg / kg / hour for the remaining 22 hours over the 24 hours starting concomitantly with each cyclophosphamide dose.

[0401] In some embodiments, the lymphodepletion comprises the step of treating the patient with an IL-2 regimen starting on the day after administration of the third population of TILs to the patient.

[0402] In some embodiments, the lymphodepletion comprises the step of treating the patient with an IL-2 regimen starting on the same day as administration of the third population of TILs to the patient.

[0403] In some embodiments, the lymphodeplete comprises 5 days of preconditioning treatment. In some embodiments, the days are indicated as days -5 through -1, or Day 0 through Day 4. In some embodiments, the regimen comprises cyclophosphamide on days -5 and -4 (i.e., days 0 and 1). In some embodiments, the regimen comprises intravenous cyclophosphamide on days -5 and -4 (i.e., days 0 and 1). In some embodiments, the regimen comprises 60 mg / kg intravenous cyclophosphamide on days -5 and -4 (i.e., days 0 and 1). In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the regimen further comprises fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine. In some embodiments, the regimen further comprises 25 mg / m2 intravenous fludarabine. In some embodiments, the regimen further comprises 25 mg / m2 intravenous fludarabine on days -5 and -1 (i.e., days 0 through 4). In some embodiments, the regimen further comprises 25 mg / m2 intravenous fludarabine on days -5 and -1 (i.e., days 0 through 4).

[0404] 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 five days.

[0405] 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.

[0406] 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 three days

[0407] 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.

[0408] 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 one day.

[0409] 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 three days.

[0410] 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.

[0411] In some embodiments, the TIL infusion used with the foregoing embodiments of myeloablative lymphodepletion regimens may be any TIL composition described herein, as well as the addition of IL-2 regimens and administration of co-therapies (such as PD-1 and PD-L1 inhibitors) as described herein.B. IL-2 Regimens

[0412] In some embodiments, the IL-2 regimen comprises a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises aldesleukin, or a biosimilar or variant thereof, administered intravenously starting on the day after administering a therapeutically effective portion of the therapeutic population of TILs, wherein the aldesleukin or a biosimilar or variant thereof is administered at a dose of 0.037 mg / kg or 0.044 mg / kg lU / kg (patient body mass) using 15-minute bolus intravenous infusions every eight hours until tolerance, for a maximum of 14 doses. Following 9 days of rest, this schedule may be repeated for another 14 doses, for a maximum of 28 doses in total. In some embodiments, IL-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In some embodiments, IL-2 is administered at a maximum dosage of up to 6 doses.

[0413] In some embodiments, the IL-2 regimen comprises a decrescendo IL-2 regimen. Decrescendo IL-2 regimens have been described in O'Day, et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporatedherein by reference. In some embodiments, a decrescendo IL-2 regimen comprises 18 x 106IU / m2 aldesleukin, or a biosimilar or variant thereof, administered intravenously over 6 hours, followed by 18 x 106 IU / m2 administered intravenously over 12 hours, followed by 18 x 106 IU / m2 administered intravenously over 24 hours, followed by 4.5 x 106 IU / m2 administered intravenously over 72 hours. This treatment cycle may be repeated every 28 days for a maximum of four cycles. In some embodiments, a decrescendo IL-2 regimen comprises 18,000,000 IU / m2 on day 1, 9,000,000 I U / m2 on day 2, and 4,500,000 IU / m2 on days 3 and 4.

[0414] In some embodiments, the IL-2 regimen comprises a low-dose IL-2 regimen. Any low- dose IL-2 regimen known in the art may be used, including the low-dose IL-2 regimens described in Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann, et al., Lancet Diabetes Endocrinol. 2013, 1, 295-305; and Rosenzwaig, et al., Ann. Rheum. Dis. 2019, 78, 209-217, the disclosures of which are incorporated herein by reference. In some embodiments, a low-dose IL-2 regimen comprises 18 x 106 IU per m2 of aldesleukin, or a biosimilar or variant thereof, per 24 hours, administered as a continuous infusion for 5 days, followed by 2-6 days without IL-2 ther...

Claims

1. WHAT IS CLAIMED IS:

1. A method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); and c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate the TIL product enriched with NARTs.

2. The method of claim 1, wherein profiling the plurality of TIL populations comprises quantification of T cell DNA copy number of the plurality of TIL populations.

3. The method of claim 2, wherein quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelica lly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells.

4. The method of any one of claims 1-3, wherein profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations.

5. The method of claim 4, wherein the panel of target genes comprises one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19.

6. The method of claims 5 or 6, wherein measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes.

7. The method of any one of claims 1-6, wherein the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI).

8. The method of claim 7, wherein the ICI comprises a CD38 inhibitor, a CTLA-4 inhibitor and / or a PD-1 / PD-L1 inhibitor.

9. The method of any one of claims 1-8, wherein the first cell culture medium in step (c)further comprises soluble IL-12 and / or a 4-1BB agonist.

10. The method of claim 9, wherein the 4-1BB agonist is urelumab.

11. The method of any one of claims 1-10, further comprising selecting CD137+, CD200+ and / or CD103+ TILs from the TIL product enriched with NARTs in step (c) to generate a positively-selected TIL product.

12. The method of any one of claims 1-11, further comprising removing PD-1H|TILs from the TIL product enriched with NARTs in step (c) or the positively-selected TIL product to generate a bystander-depleted TIL product.

13. The method of any one of claims 1-12, further comprising: d) culturing the TIL product in a second cell culture medium in the presence of artificial APCs (aAPCs).

14. The method of claim 13, wherein the aAPCs are acellular-based or cellular-based.

15. The method of claim 13 or 14, wherein the second cell culture medium comprises IL-15 and IL-21.

16. The method of any one of claims 13-15, wherein the second cell culture medium comprises L-arginine.

17. The method of claim 16, wherein the L-arginine is present at a concentration of about 5 mM.

18. The method of any one of claims 13-17, wherein the second cell culture medium comprises an NAD+ booster.

19. The method of claim 18, wherein the NAD+ booster is one or more of L-Trp, NR, NMN, NAD+, NAM, or P7C3 activator.

20. The method of any one of claims 13-19, wherein the second cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L-lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate (3-OHB), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate.

21. The method of any one of claims 13-20, wherein the second cell culture medium comprises a mitophagy activator.

22. The method of claim 21, wherein the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

23. The method of any one of claims 1-22, wherein step (a) comprises fractioning the tumor sample into multiple tumor fragments.

24. The method of claim 23, wherein the tumor sample is fractioned into 2-100 tumor fragments.

25. The method of any one of claims 1-22, wherein step (a) comprises digesting the tumor sample into a tumor digest.

26. A method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) selecting CD137+, CD200+ and / or CD103+ TILs from the first population of TILs to generate a second population of TILs; and e) Culturing the second population of TILs in a second cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

27. The method of claim 26, wherein profiling the plurality of TIL populations comprises quantification of T cell DNA copy number of the plurality of TIL populations.

28. The method of claim 27 , wherein quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelica lly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells.

29. The method of any one of claims 26-28, wherein profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations.

30. The method of claim 29, wherein the panel of target genes comprises one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19.

31. The method of claims 29 or 30, wherein measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes.

32. The method of any one of claims 26-31, wherein the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI).

33. The method of claim 32, wherein the ICI comprises a CD38 inhibitor, a CTLA-4 inhibitor and / or a PD-1 / PD-L1 inhibitor.

34. The method of any one of claims 26-33, wherein the first cell culture medium in step (c) further comprises soluble IL-12 and / or a 4-1BB agonist.

35. The method of claim 34, wherein the 4-1BB agonist is urelumab.

36. The method of any one of claims 26-35, further comprising removing PD-1H|TILs from the first or second population of TILs to generate a bystander-depleted TIL product.

37. The method of any one of claims 26-36, wherein the aAPCs are acellular-based or cellular-based.

38. The method of any one of claims 26-37, wherein the second cell culture medium comprises IL-15 and IL-21.

39. The method of any one of claims 26-38, wherein the second cell culture medium comprises L-arginine.

40. The method of claim 39, wherein the L-arginine is present at a concentration of about 5 mM.

41. The method of any one of claims 26-40, wherein the second cell culture medium comprises an NAD+ booster.

42. The method of claim 41, wherein the NAD+ booster is one or more of L-Trp, NR, NMN, NAD+, NAM, or P7C3 activator.

43. The method of any one of claims 26-42, wherein the second cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L-lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate (£-OH B), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate.

44. The method of any one of claims 26-43, wherein the second cell culture medium comprises a mitophagy activator.

45. The method of claim 44, wherein the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

46. The method of any one of claims 26-45, wherein step (a) comprises fractioning the tumor sample into multiple tumor fragments.

47. The method of claim 46, wherein the tumor sample is fractioned into 2-100 tumor fragments.

48. The method of any one of claims 26-47, wherein step (a) comprises digesting the tumor sample into a tumor digest.

49. A method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling the plurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) Culturing the first population of TILs in a second cell culture medium to generate a second population of TILs; and e) Culturing the second population of TILs in a third cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

50. The method of claim 49, wherein profiling the plurality of TIL populations comprisesquantification of T cell DNA copy number of the plurality of TIL populations.

51. The method of claim 50, wherein quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelica lly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells.

52. The method of any one of claims 49-51, wherein profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations.

53. The method of claim 52, wherein the panel of target genes comprises one or more genes selected from one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19.

54. The method of claims 52 or 53, wherein measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes.

55. The method of any one of claims 49-54, wherein the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI).

56. The method of claim 55, wherein the ICI comprises a CD38 inhibitor, a CTLA-4 inhibitor and / or a PD-1 / PD-L1 inhibitor.

57. The method of any one of claims 49-56, wherein the first cell culture medium in step (c) further comprises soluble IL-12 and / or a 4-1BB agonist.

58. The method of claim 57, wherein the 4-1BB agonist is urelumab.

59. The method of any one of claims 49-58, further comprising selecting CD137+, CD200+ and / or CD103+ TILs from the first or second population of TILs to generate a positively- selected TIL product.

60. The method of any one of claims 49-59, further comprising removing PD-1H|TILs from the first or second population of TILs or the positively-selected TIL product to generate a bystander-depleted TIL product.

61. The method of any one of claims 49-60, wherein the aAPCs are acellular-based or cellular-based.

62. The method of any one of claims 49-61, wherein the third cell culture medium comprises IL-15 and IL-21.

63. The method of any one of claims 49-62, wherein the third cell culture medium comprises L-arginine.

64. The method of claim 63, wherein the L-arginine is present at a concentration of about 5 mM.

65. The method of any one of claims 49-64, wherein the third cell culture medium comprises an NAD+ booster.

66. The method of claim 65, wherein the NAD+ booster is selected from one or more of L- Trp, NR, NMN, NAD+, NAM, or P7C3 activator.

67. The method of any one of claims 49-66, wherein the third cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L-lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate (|3-OHB), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate.

68. The method of any one of claims 49-67, wherein the third cell culture medium comprises a mitophagy activator.

69. The method of claim 68, wherein the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

70. The method of any one of claims 49-69, wherein step (a) comprises fractioning the tumor sample into multiple tumor fragments.

71. The method of claim 70, wherein the tumor sample is fractioned into 2-100 tumor fragments.

72. The method of any one of claims 49-69, wherein step (a) comprises digesting the tumor sample into a tumor digest.

73. A method for making a tumor infiltrating lymphocytes (TILs) product enriched with neoantigen-reactive T cells (NARTs), comprising: a) Generating a plurality of TIL populations by fractioning a tumor sample; b) Selecting one or more TIL populations enriched with NARTs by profiling theplurality of TIL populations using multiplex digital PCR (dPCR); c) Preferentially expanding the NARTs by co-culturing the one or more TIL populations enriched with NARTs with tumor cells from the tumor sample in a first cell culture medium to generate a first population of TILs; d) Culturing the first population of TILs in a second cell culture medium to generate a second population of TILs; e) selecting CD137+, CD200+ and / or CD103+ TILs from the second population of TILs to generate a third population of TILs; and f) Culturing the third population of TILs in a third cell culture medium in the presence of artificial APCs (aAPCs) to generate the TIL product enriched with NARTs.

74. The method of claim 73, wherein profiling the plurality of TIL populations comprises quantification of T cell DNA copy number of the plurality of TIL populations.

75. The method of claim 74, wherein quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is bial lelica lly absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells.

76. The method of any one of claims 73-75, wherein profiling the plurality of TIL populations comprises measurement of the expression level of a panel of target genes of the plurality of TIL populations.

77. The method of claim 76, wherein the panel of target genes comprises one or more genes selected from one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19.

78. The method of claims 75 or 76, wherein measurement of the expression level of a panel of target genes comprises quantification of the mRNA of the target genes.

79. The method of any one of claims 73-78, wherein the first cell culture medium in step (c) comprises an immune checkpoint inhibitor (ICI).

80. The method of claim 79, wherein the ICI comprises a CTLA-4 inhibitor and / or a PD-l / PD- L1 inhibitor.

81. The method of any one of claims 73-80, wherein the first cell culture medium in step (c) further comprises soluble IL-12 and / or a 4-1BB agonist.

82. The method of claim 81, wherein the 4-1BB agonist is urelumab.

83. The method of any one of claims 73-82, further comprising removing PD-lHiTILs from the first, second or third population of TILs to generate a bystander-depleted TIL product.

84. The method of any one of claims 73-83, wherein the aAPCs are acellular-based or cellular-based.

85. The method of any one of claims 73-84, wherein the third cell culture medium comprises IL-15 and IL-21.

86. The method of any one of claims 73-85, wherein the third cell culture medium comprises L-arginine.

87. The method of claim 86, wherein the L-arginine is present at a concentration of about 5 mM.

88. The method of any one of claims 73-87, wherein the third cell culture medium comprises an NAD+ booster.

89. The method of claim 88, wherein the NAD+ booster is selected from one or more of L- Trp, NR, NMN, NAD+, NAM, or P7C3 activator.

90. The method of any one of claims 73-89, wherein the third cell culture medium comprises 1.5-3 mM glucose, 0.5-5 mM Na-L-lactate, 1-5 mM short chain fatty acids Beta-hydroxy butyrate (3-OHB), 0.5 mM Sodium butyrate, 0.1-1 mM Sodium Propionate, and / or 1-10 mM Sodium Acetate.

91. The method of any one of claims 73-90, wherein the third cell culture medium comprises a mitophagy activator.

92. The method of claim 91, wherein the mitophagy activator comprises metformin, valproic acid, or Urolithin A.

93. The method of any one of claims 73-92, wherein step (a) comprises fractioning the tumor sample into multiple tumor fragments.

94. The method of claim 93, wherein the tumor sample is fractioned into 2-100 tumorfragments.

95. The method of any one of claims 73-92, wherein step (a) comprises digesting the tumor sample into a tumor digest.

96. A method for assessing tumor reactivity of a population of T cells, comprising measurement of the expression level of a panel of target genes of the population of T cells.

97. The method of claim 96, wherein the panel of target genes comprises one or more genes selected from one or more of CXCL13, CD200, TRBC, YWHAZ, CD20, or CD19.

98. The method of claims 96 or 97, wherein measurement of the expression level of the panel of target genes comprises quantification of the mRNA molecules of each of the target genes.

99. The method of claim 98, wherein quantification of the mRNA molecules of each of the target genes comprises determining the copy number of the mRNA molecules of each of the target genes.

100. The method of claim 99, wherein determining the copy number of the mRNA molecules comprises reverse transcription of the mRNA molecules into cDNA molecules.

101. The method of claim 100, wherein the cDNA molecules are quantified by digital PCR (dPCR).

102. The method of any one of claims 96-101, wherein the population of T cells is admixed with a population of non-T cells.

103. The method of claim 102, wherein the non-T cells are tumor cells.

104. The method of claim 102 or 103, further comprising quantification of T cell DNA copy number of the population of T cells.

105. The method of claim 104, wherein quantification of T cell DNA copy number comprises detection of a unique T cell DNA marker that is biallelically absent in T cells due to VDJ rearrangements, a regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells, and a reference DNA marker existing as a diploid in all cells.

106. The method of claim 105, wherein the unique T cell DNA marker that is biallelically absent in T cells due to VDJ rearrangements is TRAB, the regional corrector that allows correction of possible copy number alterations at the T cell marker locus in cancer cells is TRBC, and the reference DNA marker existing as a diploid in all cells is RPP30.

107. The method of any one of claims 96-106, wherein the T cells are tumor infiltrating lymphocytes (TILs).