Method for assessing proliferative potency of genetically edited t cells

Abstract

The present invention provides improved methods for assessing the proliferative efficacy of genetically edited T cells, such as genetically edited tumor infiltrating lymphocytes (TILs), CART cells, and the like.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 384,598, filed on November 21, 2022, the entire content of which is incorporated herein by reference in its entirety. Background of the Invention

[0003] TALEN gene editing has been shown to permanently disrupt genes in human and murine T cells with high specificity. However, if TALEN remains active over an extended period, off - target cleavage may occur. These ectopic modifications within cells can contribute to cell transformation. Interleukin 2 (IL - 2) is a cytokine required for T - cell proliferation. IL - 2 - independent proliferation would indicate cell transformation and could be considered cancerous.

[0004] The present invention provides a shortened process and method for evaluating the proliferative potency of gene - edited T cells, such as tumor - infiltrating lymphocytes (TIL) and chimeric antigen receptor T (CART) cells. Summary of the Invention

[0005] Provided herein is an improved method for evaluating the proliferative potency of gene - edited T cells, such as gene - edited tumor - infiltrating lymphocytes (TIL), chimeric antigen receptor T (CART) cells, etc.

[0006] In some embodiments, provided herein is a method for evaluating the proliferation index of a population of gene - edited T cells, the method comprising: (a) culturing a first portion of the population of gene - edited T cells in a cell culture medium without IL - 2; (b) adding an agent to the cell culture medium, wherein the agent is incorporated into the DNA of the cells during proliferation; (c) measuring the amount of the agent incorporated into the gene - edited T cells at one or more time points; and (d) calculating the proliferation index of the population of gene - edited T cells based on the amount of the agent incorporated into the gene - edited T cells at one or more time points.

[0007] In some embodiments, the agent is a modified nucleotide selected from the group consisting of: EdU (5 - ethynyl - 2′ - deoxyuridine), 5 - ethynyluridine (5 - EU), F - ara - EdU, bromo - 2′ - deoxyuridine (BrdU), and 3 H] thymidine ( 3H]TdR). In some embodiments, the agent is EdU. In some embodiments, measuring the amount of the agent incorporated into T cells includes performing a click reaction using HRP. In some embodiments, the method further includes adding Amplex UltraRed reagent, wherein Amplex UltraRed is converted by HRP into a fluorescent product. In some embodiments, the method further includes measuring the amount of the fluorescent product using a fluorescence reader. In some embodiments, one or more time points are selected from the group consisting of day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, and day 28. In some embodiments, one or more time points include day 1, day 7, day 10, and day 14. In some embodiments, the agent is added on day 0, day 6, day 9, and / or day 13. In some embodiments, the agent is added 24 hours before measuring the amount of the agent incorporated into the gene-edited T cells. In some embodiments, the agent is added at 0 uM, 10 uM, 20 uM, and / or 40 uM. In some embodiments, the method further includes culturing a second portion of the gene-edited T cell population in cell culture medium having IL-2 as a positive control. In some embodiments, the method further includes culturing the transformed T cell population in cell culture medium without IL-2 as a positive control. In some embodiments, the transformed T cells are Jurkat cells.

[0008] In some embodiments, the gene-edited T cells are gene-edited tumor-infiltrating lymphocytes (TILs) or CAR T cells. In some embodiments, the gene-edited T cells are gene-edited TILs. In some embodiments, the gene-edited TILs comprise a TALE nuclease system for regulating the expression of at least one protein. In some embodiments, the TALE nuclease system regulates the expression of PD-1. In some embodiments, the TALE nuclease system regulates the expression of CTLA-4. In some embodiments, the TALE nuclease system regulates the expression of LAG-3. In some embodiments, the TALE nuclease system regulates the expression of CISH. In some embodiments, the TALE nuclease system regulates the expression of CBL-B. In some embodiments, the TALE nuclease system regulates the expression of TIGIT. In some embodiments, the gene-edited TILs include a first TALE nuclease system for regulating the expression of a first protein and a second TALE nuclease system for regulating the expression of a second protein. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and / or CBL-B. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CTLA-4. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and LAG-3. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CISH. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CBL-B. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and TIGIT. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and LAG-3. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CISH. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CBL-B. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CISH. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CBL-B. In some embodiments, the first TALE nuclease system and the second TALE nuclease system regulate the expression of CISH and CBL-B. In some embodiments, the gene-edited T cells are CAR T cells. In some embodiments,The CAR of the CAR-T cells is specific for an antigen selected from the group consisting of: CD19; CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule 1, CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B-cell maturation antigen (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin 13 receptor subunit α-2; mesothelin; interleukin 11 receptor α (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen 4 (SSEA-4); CD20; folate receptor α; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme; prostate acid phosphatase (PAP); mutant elongation factor 2 (ELF2M); ephrin B2; fibroblast activation protein α (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogenic fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog (Abl); tyrosinase; ephrin A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5,Member D (GPRC5D); Chromosome X open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide moiety of globo H glycosphingolipid (GloboH); Breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); Adrenergic receptor beta-3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); Spermatid protein 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein p53 (p53); p53 mutant; Prostein; Survivin; Telomerase; Prostate cancer tumor antigen 1; Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); Paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen recognized by T cells 3 (SART3); Paired box protein Pax-5 (PAX5); Proacrosomal protein-binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase (LCK); Kinase anchor protein 4 (AKAP-4); Synovial sarcoma,X breakpoint 2 (SSX2); receptor for advanced glycation end products-1 (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican 3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0009] The present disclosure further provides a method for treating a cancer patient, the method comprising: (a) generating a gene-edited TIL population from a tumor excised from a cancer patient; (b) evaluating the proliferation index of the gene-edited TIL population using the method according to any one of claims 1 to 36; and (c) administering a therapeutically effective dose of the gene-edited TIL to the cancer patient if the proliferation index of the gene-edited TIL population is lower than the proliferation index of a positive control.

[0010] In some embodiments, the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancers caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma. In some embodiments, the therapeutically effective dose of the gene-edited TIL is about 1×10 9 to about 1×10 11a TIL. In some embodiments, a non-myeloablative lymphodepletion regimen has been administered to the patient prior to administering a therapeutically effective dose of the gene-edited TIL to the patient in step (c). In some embodiments, the method further comprises the step of treating the patient with a high-dose IL-2 regimen starting on the day after administering a therapeutically effective dose of the gene-edited TIL to the patient in step (c). In some embodiments, the cancer is melanoma. In some embodiments, the cancer is metastatic melanoma. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is metastatic NSCLC. In some embodiments, the gene editing causes silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the gene-edited TIL population. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A - Figure 1B : Exemplary manufacturing process of gene-edited TIL.

[0012] Figure 2 : Time course of fold expansion of TIL in the presence and absence of IL-2.

[0013] Figure 3 : Proliferation characteristics of Jurkat cells at various seeding densities, multiple time points (day 1, day 5, and day 7), and multiple EdU concentrations.

[0014] Figure 4 : Proliferation characteristics of Jurkat cells at various seeding densities and multiple EdU addition intervals.

[0015] Figure 5 : Proliferation characteristics of two gene-edited TIL batches at multiple time points (day 1, day 7, day 10, and day 14).

[0016] Figure 6 : Proliferation characteristics of five gene-edited TIL batches at multiple time points (day 7 and day 10).

[0017] BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0018] SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

[0019] SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

[0020] SEQ ID NO:3 is the amino acid sequence of recombinant human IL-2 protein.

[0021] SEQ ID NO:4 is the amino acid sequence of aldesleukin.

[0022] SEQ ID NO:5 is an IL-2 form.

[0023] SEQ ID NO:6 is the amino acid sequence of nemvaleukin alfa.

[0024] SEQ ID NO:7 is an IL-2 form.

[0025] SEQ ID NO:8 is a mucin domain polypeptide.

[0026] SEQ ID NO:9 is the amino acid sequence of recombinant human IL-4 protein.

[0027] SEQ ID NO:10 is the amino acid sequence of recombinant human IL-7 protein.

[0028] SEQ ID NO:11 is the amino acid sequence of recombinant human IL-15 protein.

[0029] SEQ ID NO:12 is the amino acid sequence of recombinant human IL-21 protein.

[0030] SEQ ID NO:13 is the target PD-1 sequence.

[0031] SEQ ID NO:14 is the target PD-1 sequence.

[0032] SEQ ID NO:15 is the repeated PD-1 left repeat sequence.

[0033] SEQ ID NO:16 is the repeated PD-1 right repeat sequence.

[0034] SEQ ID NO:17 is the repeated PD-1 left repeat sequence.

[0035] SEQ ID NO:18 is the repeated PD-1 right repeat sequence.

[0036] SEQ ID NO:19 is the PD-1 left TALEN nuclease sequence.

[0037] SEQ ID NO:20 is the PD-1 right TALEN nuclease sequence.

[0038] SEQ ID NO:21 is the PD-1 left TALEN nuclease sequence.

[0039] SEQ ID NO:22 is the PD-1 right TALEN nuclease sequence.

[0040] SEQ ID NO:23 is the exemplary Clo05 l nuclease domain amino acid sequence.

[0041] I. Definitions

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited herein are incorporated by reference in their entirety.

[0043] As used herein, the terms "co-administration", "co-administering", "administered in combination with", "administering in combination with", "simultaneous", and "concurrent" encompass the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, e.g., multiple TILs) to a subject such that the two active pharmaceutical ingredients and / or their metabolites are present in the subject's body at the same time. Co-administration includes administration simultaneously 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. Administration simultaneously in separate compositions and administration in a composition in which two agents are present are preferred.

[0044] The term "in vivo" refers to an event that occurs within the body of a subject.

[0045] The term "in vitro" refers to an event that occurs outside the body of a subject. In vitro assays encompass cell-based assays in which live or dead cells are employed and also cell-free assays in which intact cells are not employed.

[0046] The term "ex vivo" refers to an event that involves the processing or performance of a procedure on cells, tissues, and / or organs that have been removed from the body of a subject. Optionally, the cells, tissues, and / or organs can be returned to the body of the subject by surgical or therapeutic means.

[0047] The term "rapid expansion" means that the number of antigen-specific TILs increases by at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) within a one-week period, more preferably by at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) within a one-week period, or most preferably by at least about 100-fold within a one-week period. Numerous rapid expansion protocols are described herein.

[0048] "Tumor infiltrating lymphocytes" or "TIL" in this text refers to a population of cells initially obtained as white blood cells that have left the subject's blood and migrated to the tumor. TIL includes, but is not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + T cells, natural killer cells, dendritic cells, and M1 macrophages. TIL includes both primary TIL and secondary TIL. "Primary TIL" are those TIL obtained from patient tissue samples as outlined herein (sometimes referred to as "fresh harvested"), and "secondary TIL" are any population of TIL cells that have been expanded or proliferated as discussed herein, including but not limited to bulk TIL and expanded TIL ("REP TIL" or "post-REP TIL"). TIL cell populations can include genetically modified TIL.

[0049] "Population of cells" (including TIL) in this text refers to many cells that share a common characteristic. Generally, the number of the population typically ranges from 1X10 6 to 1X10 10 , and different TIL populations contain different numbers. For example, in the presence of IL-2, the initial growth of primary TIL produces a bulk TIL population of approximately 1×10 8 cells. REP expansion is generally for providing a population of 1.5×10 9 to 1.5×10 10 cells for infusion.

[0050] "Cryopreserved TIL" in this text refers to TIL (primary, bulk, or expanded (REP TIL)) that are processed and stored in the range of approximately -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, "cryopreserved TIL" is distinguished from frozen tissue samples that can serve as a source of primary TIL.

[0051] "Thawed cryopreserved TIL" in this text refers to a population of TIL that have been previously cryopreserved and then processed to be restored to room temperature or higher temperature (including but not limited to cell culture temperature or a temperature at which TIL can be administered to a patient).

[0052] TILs can generally be biochemically defined using cell surface markers or can be functionally defined by their ability to infiltrate tumors and affect treatment. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after being reintroduced into a patient.

[0053] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% DMSO to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to a cryopreservation medium obtained from Stemcell Technologies or from Biolife Solutions. The CS10 medium may be referred to by the trade name " CS10". The CS10 medium is a serum-free, animal component-free medium containing DMSO. In some embodiments, the CS10 medium contains 10% DMSO.

[0054] As used herein, the terms "fragmentation", "fragment", and "fragmented" are used to describe the process for disrupting a tumor, including mechanical fragmentation methods such as crushing, slicing, dissecting, and chopping tumor tissue, and any other method for disrupting the physical structure of tumor tissue.

[0055] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0056] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from a donor's whole blood or apheresis product. In some embodiments, PBLs are isolated from a donor's whole blood or apheresis product by positive or negative selection for a T cell phenotype such as a CD3+CD45+ T cell phenotype.

[0057] The term "anti-CD3 antibody" refers to an antibody or variant thereof that targets the CD3 receptor in the T-cell antigen receptor of mature T cells, e.g., a monoclonal antibody and the antibody includes a human antibody, humanized antibody, chimeric antibody, or murine antibody. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0058] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof that targets the CD3 receptor in the T-cell antigen receptor of mature T cells, including a human antibody, humanized antibody, chimeric antibody, or murine antibody, and includes a commercially available form such as OKT-3 (30 ng / mL, pure MACS GMP CD3, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or a variant, conservative amino acid substitution, glycosylation form, or biosimilar. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). The hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. The hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned the catalogue number 86022706.

[0059] Table 1. Amino acid sequences of muromonab (exemplary OKT-3 antibody).

[0060]

[0061] 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, which forms include human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 applicable to the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses recombinant forms of human IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at a price of 22 million IU per single-use vial), and recombinant IL-2 forms commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-TanyTechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-209-b) and other commercial equivalents from other suppliers. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin applicable to the present invention is given in Table 2 (SEQ ID NO: 4).

[0062] Table 2. Amino acid sequences of interleukins.

[0063]

[0064]

[0065] 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 (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. After activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression and induces B cell class switching to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from a number of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 9).

[0066] The term "IL-7" (also referred to herein as "IL7") refers to the glycosylated tissue-derived cytokine known as interleukin 7, which is obtainable from stromal cells and epithelial cells and from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 stimulates T cell development. IL-7 binds to the IL-7 receptor, which is a heterodimer composed of the IL-7 receptor α and the common γ chain receptor and which sends a series of signals important for intrathymic T cell development and peripheral survival. Recombinant human IL-7 suitable for use in the present invention is commercially available from a number of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 10).

[0067] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15 and includes all forms of IL-2, which forms include human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain that contains 114 amino acids (and an N-terminal methionine) and has a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from a number of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is given in Table 2 (SEQ ID NO:11).

[0068] 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, which forms include human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. 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 that contains 132 amino acids and has a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from a number of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is given in Table 2 (SEQ ID NO:12).

[0069] When referring to an "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount", the exact amount of the composition of the present invention to be administered can be determined by a physician considering the age, weight, tumor size, degree of infection or metastasis, and individual differences in the condition of the patient (subject). Generally, it can be stated that a pharmaceutical composition comprising the tumor-infiltrating lymphocytes described herein (e.g., secondary TIL or genetically modified cytotoxic lymphocytes) can be administered at a dose of 10 4 to 10 11 cells / kg body weight (e.g., 10 5 to 10 6 , 10 5 to 10 10 , 10 5 to 10 11 , 10 6 to 10 10 , 10 6 to 10 11 , 10 7 to 10 11 , 10 7 to 10 10 , 10 8 to 10 11 , 10 8 to 10 10 , 10 9 to 10 11 or 10 9 to 10 10 cells / kg body weight), including all integer values within these ranges. The TIL (including genetically modified cytotoxic lymphocytes in some cases) compositions can also be administered multiple times at these doses. TIL (including genetically engineered TIL in some cases) can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). By monitoring the signs of the patient's disease and adjusting the treatment accordingly, a person skilled in the medical art can readily determine the optimal dose and treatment regimen for a particular patient.

[0070] The term "hematological malignancy", "hematologic malignancy", or terms of related meaning refers to cancers and tumors of the hematopoietic and lymphoid tissues of a mammal, 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 lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin lymphoma. The term "B-cell hematological malignancy" refers to a hematological malignancy that affects B cells.

[0071] The term "liquid tumor" refers to an abnormal cell mass that is liquid in nature. Liquid tumor cancers include but are not limited to leukemia, myeloma, and lymphoma, 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 in the tissue type of the cell source.

[0072] As used herein, the term "microenvironment" may refer to an entity as a whole or the blood tumor microenvironment or an individual subset of cells within the microenvironment. As used herein, the tumor microenvironment refers to "a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promotes tumor transformation, supports tumor growth and invasion, protects the tumor from the host immune system, promotes treatment resistance, and provides a niche for the growth of metastatic seeding", 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 due to the immunosuppressive nature of the microenvironment.

[0073] In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TIL according to the present invention. In some embodiments, a TIL population may be provided, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TIL according to the present invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide at 60 mg / kg / d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine at 25 mg / m2 / d for 5 days (days 27 to 23 prior to TIL infusion). In some embodiments, after non-myeloablative chemotherapy is performed and TIL infusion is performed according to the present invention (on day 0), the patient receives an intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours until physiological tolerance is reached.

[0074] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing the therapeutic effect by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes also referred to as "immunosuppressive conditioning") on the patient prior to introduction of the TIL of the present invention.

[0075] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds as described herein that is sufficient to achieve a desired application, including but not limited to the treatment of a disease. The therapeutically effective amount may vary depending on the desired application (in vitro or in vivo) or the subject and disease condition being treated (e.g., the weight, age, and sex of the subject), the severity of the disease condition, or the mode of administration. The term also applies to a dose that will induce a specific response in a target cell (e.g., reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the tissue to which it is administered, and the physical delivery system carrying the compound.

[0076] As used herein, the terms "treatment", "treating", "treat", etc. refer to obtaining a desired pharmacological and / or physiological effect. Such an effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or an adverse effect caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease in a subject who may be susceptible to the disease but has not been diagnosed as having the disease; (b) inhibiting the disease, i.e., preventing its development or progression; and (c) alleviating the disease, i.e., causing regression and / or remission of one or more symptoms of the disease. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect, even in the absence of a disease or medical condition. For example, "treatment" encompasses the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.

[0077] The terms "non-myeloablative chemotherapy", "non-myeloablative lymphodepletion", "NMALD", "NMA LD", "NMA-LD", and any variants of the foregoing may be used interchangeably to denote a chemotherapy regimen that is designed to deplete the lymphoid immune cells of a patient while avoiding depletion of the patient's myeloid immune cells. Typically, a patient has received a course of non-myeloablative chemotherapy prior to administration of tumor-infiltrating lymphocytes as described herein.

[0078] When the term "heterologous" is used to refer to a portion of a nucleic acid or protein, the term indicates that the nucleic acid or protein contains two or more subsequences that do not have the same relationship to each other in nature. For example, a nucleic acid is typically recombinantly produced and has two or more sequences from unrelated genes that are arranged together to form 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 contains two or more subsequences that do not exist in the same relationship to each other in nature (e.g., a fusion protein).

[0079] In the context of two or more nucleic acids or polypeptides, the terms "sequence identity", "percent identity", and "sequence percent identity" (or synonyms thereof, e.g., "99% identical") refer to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned (introducing gaps if necessary) to achieve maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software for obtaining alignments of amino acid or nucleotide sequences are known in the art. Suitable procedures for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. Government's National Center for Biotechnology Information. The BLASTN or BLASTP algorithms can be used to compare two sequences. 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 of ordinary skill in the art can determine the appropriate parameters for maximum alignment using a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0080] As used herein, the term "variant" encompasses, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may involve, for example, the substitution of similar charged or uncharged amino acids. The variant retains the ability to specifically bind antigen with the reference antibody. The term variant also includes polyethylene glycolated antibodies or proteins.

[0081] "Tumor infiltrating lymphocytes" or "TIL" herein means a population of cells originally obtained as white blood cells that have left the subject's blood and migrated to a tumor. TIL includes, but is not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 +T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and secondary TILs. "Primary TILs" are those TILs obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly harvested TILs"), and "secondary TILs" are any population of TIL cells that have been expanded or proliferated as discussed herein, including but not limited to large numbers of TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, secondary expanded TILs or secondary additional expanded TILs.

[0082] TILs can generally be biochemically defined using cell surface markers or can be functionally defined by their ability to infiltrate tumors and affect therapy. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, 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 after being reintroduced into a patient. TILs can be further characterized by potency – for example, if 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, then the TILs can be considered to have potency. If, for example, interferon (IFNγ) 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, then the TILs can be considered to have potency.

[0083] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, base moiety, and / or linkages between deoxyribonucleotides in an oligonucleotide.

[0084] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0085] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients (such as other drugs) may also be incorporated into the described compositions and methods.

[0086] The terms "about" and "approximately" mean within a range of values that are statistically significant. Such ranges can be within an order of magnitude, preferably within 50% of a given value or range, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The allowable deviations covered by the terms "about" or "approximately" depend on the particular system under study and can be readily understood by those of ordinary skill in the art. In addition, 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 can be approximated and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of skill in the art. Generally, whether or not explicitly stated, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are "about" or "approximately". It should be noted that very different embodiments in terms of size, shape, and dimensions may adopt the described arrangements.

[0087] The transitional terms "comprising," "consisting essentially of," and "consisting of," as used in the appended claims in their original and modified forms, define the scope of the claims, where additional claim elements or steps (if any) that are not recited are excluded from the scope of the claims. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in the latter case, excludes impurities ordinarily associated with the specified materials. The term "consisting essentially of" limits the scope of the claim to the specified elements, steps, or materials and those that do not materially affect the basic and novel characteristics of the claimed invention. In alternative embodiments, all of the compositions, methods, and kits described herein embodying the present invention can be more specifically defined by any one of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0088] The term "antibody" and its plural form "antibodies" refer to an entire immunoglobulin and any antigen-binding fragment ("antigen-binding portion") or single chain thereof. "Antibody" further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region is composed of three domains CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region is composed of one domain C L . The V H and V L regions of the antibody can be further subdivided into regions of hypervariability, which are referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), and can be interspersed with more conserved regions, called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigenic epitopes. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0089] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that, when presented by a major histocompatibility complex (MHC) molecule, can be bound by an antibody or a TCR. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen can induce a humoral immune response or a cellular immune response, resulting in the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require the antigen to contain a Th cell epitope or be linked to a Th cell epitope. An antigen may also have one or more epitopes (e.g., B epitopes and T epitopes). In some embodiments, an antigen typically reacts with its corresponding antibody or TCR in a highly specific and selective manner, preferably without reacting with a large number of other antibodies or TCRs that may be induced by other antigens.

[0090] The term "monoclonal antibody", "mAb", "monoclonal antibody composition" or plural forms thereof refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for certain receptors can be prepared using knowledge and techniques in the art by injecting a suitable antigen into a test subject and then isolating hybridomas that express antibodies having the desired sequence or functional characteristics. The DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not produce immunoglobulins (such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The recombinant production of antibodies will be described in more detail below.

[0091] As used herein, the "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of the V L domain, the V H domain, the C L domain and the CH1 domain; (ii) F(ab′)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bond at the hinge region; (iii) consisting of the V Han Fd fragment consisting of a domain and a CH1 domain; (iv) an Fv fragment consisting of the V L domain and the V H domain, (v) a domain antibody (dAb) fragment (Ward et al., Nature, 1989, 341, 544-546), which can be composed of the V H domain or the V L domain; and (vi) an isolated complementarity-determining region (CDR). In addition, although the two domains V L and V H of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker so that they can be prepared as a single protein chain, in which the V L region and the V H region pair to form a monovalent molecule, called a single-chain Fv (scFv); see, for example, Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as that of the intact antibody. In some embodiments, the scFv protein domain contains the V H portion and the V L portion. If the V L domain is the N-terminal portion of the scFv molecule, the scFv molecule is designated V L -L-V H ; or if the V H domain is the N-terminal portion of the scFv molecule, the scFv molecule is designated V H -L-V L . Methods for preparing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692, U.S. Patent 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 patents and documents are incorporated herein by reference.

[0092] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework regions and the CDRs are derived from human germline immunoglobulin sequences. In addition, if an antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0093] The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable regions in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene that are fused to immortalized cells.

[0094] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal human immunoglobulin gene animals (such as mice) or hybridomas prepared therefrom (described further below), (b) antibodies isolated from host cells transformed to express human antibodies (e.g., from transfectomas), (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, in vivo somatic mutagenesis), such that the amino acid sequences of the V H region and the V L region are sequences that, although derived from and related to human germline V H sequences and V L sequences, may not naturally occur in the in vivo human antibody germline repertoire.

[0095] As used herein, "isotype" refers to the class of antibody (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0096] The phrases "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds an antigen".

[0097] The term "human antibody derivative" refers to any modified form of a human antibody, including conjugates of an antibody with another active pharmaceutical ingredient or an antibody. The terms "conjugate", "antibody-drug conjugate", "ADC" or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0098] The terms "humanized antibody", "humanized antibodies", and "humanized" are intended to refer to antibodies in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto a human framework sequence. Additional framework region modifications can be made within the human framework sequence. A humanized form of a non-human (e.g., murine) antibody is a chimeric antibody that contains the minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues from the recipient hypervariable regions are replaced with the residues from the hypervariable regions of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. In addition, a humanized antibody may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will contain substantially all of at least one and usually two variable domains, where all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those of the human immunoglobulin sequence. A humanized antibody optionally will also contain at least a portion of the immunoglobulin constant region (Fc), usually at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to incorporate any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants can include, for example, any of the amino acid substitutions disclosed in the following applications: International Patent Application Publication Nos. WO1988 / 07089A1, WO 1996 / 14339A1, WO 1998 / 05787A1, WO 1998 / 23289A1, WO 1999 / 51642A1, WO 99 / 58572A1, WO 2000 / 09560A2, WO 2000 / 32767A1, WO 2000 / 42072A2, WO 2002 / 44215A2, WO 2002 / 060919 A2, WO 2003 / 074569A2, WO 2004 / 016750A2, WO 2004 / 029207A2, WO 2004 / 035752A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO2004 / 099249A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981A2, WO 2005 / 092925 A2, WO 2005 / 123780A2, WO 2006 / 019447A1, 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 herein by reference.

[0099] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a murine antibody and the constant region sequence is derived from a human antibody.

[0100] "Diabody" is a small antibody fragment having two antigen-binding sites. The fragment contains a heavy chain variable domain (V L ) linked to a light chain variable domain (V H ) in the same polypeptide chain (V H -V L or V L -V H)。By using an overly short linker, pairing between two domains on the same chain is allowed, forcing said domains to pair with complementary domains on another chain and generating two antigen-binding sites. Bispecific antibodies are described in more detail, for example, in European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0101] The term "glycosylation" refers to modified derivatives of antibodies. Non-glycosylated antibodies lack glycosylation. For example, glycosylation can be altered to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Non-glycosylation can increase the affinity of an antibody for an antigen, as described in U.S. Patent Nos. 5,714,350 and 6,350,861. Additionally or alternatively, antibodies can be prepared with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an 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 the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, for example, 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 that encodes fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating the enzyme associated with the α1,6 bond, and also describes cell lines with low enzyme activity (for adding fucose to N-acetylglucosamine that binds to the Fc region of the antibody) or no enzyme activity, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO 03 / 035835 describes a variant CHO cell line, the Lec 13 cell, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in this 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 a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNAc structures, which result in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosyl residues of an antibody can be removed using a fucosidase. For example, the fucosidase α-L-fucosidase removes fucosyl residues from an antibody, as described in Tarentino et al., Biochem. 1975, 14, 5516-5523).

[0102] "PEGylation" refers to the reaction of a modified antibody or fragment thereof with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. For example, PEGylation can increase the biological (e.g., serum) half-life of an antibody. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(C1-C 10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be PEGylated can be a non-glycosylated antibody. Methods for PEGylation are known in the art and can be applied to the antibodies of the present invention, as described, for example, in European Patent Nos. EP 0154316 and EP 0401384 and U.S. Patent No. 5,824,778, the disclosures of which are incorporated herein by reference in their entirety).

[0103] The term "biosimilar" means a biological product, including a monoclonal antibody or protein, that is highly similar to a reference biological product licensed in the United States, notwithstanding minor differences in clinically inactive components, and 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. Additionally, a similar biological medicine or "biosimilar" is a biological medicine that is similar to another biological medicine that has been authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions. A biological product or biological medicine is a medicine prepared from or derived from a biological source, such as bacteria 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), then the protein of reference aldesleukin approved by the drug regulatory agency is a "biosimilar" of aldesleukin or "its biosimilar" of aldesleukin. In Europe, a similar biological medicine or "biosimilar" is a biological medicine that is similar to another biological medicine that has been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for the application of similar biological medicines in Europe is Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC as amended, and thus in Europe, under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, biosimilars can be authorized, authorized for approval, or applied for authorization. An authorized originator biological medicine can be referred to as a "reference medicine" in Europe. Some requirements for considering a product as a biosimilar are listed in the CHMP guidelines on similar biological medicines. Additionally, the EMA also provides specific product guidelines according to specific products, including guidelines related to monoclonal antibody biosimilars, and publishes them on its website. The biosimilars described herein may be similar to the reference medicine in terms of quality characteristics, biological activity, mechanism of action, safety profiles, and / or efficacy. Additionally, biosimilars can be used or are intended to be used to treat the same disorders as the reference medicine. Therefore, the biosimilars described herein can be considered to have quality characteristics similar or highly similar to those of the reference medicine. Alternatively, or in addition, the biosimilars described herein can be considered to have biological activity similar or highly similar to that of the reference medicine. Alternatively, or in addition, the biosimilars described herein can be considered to have safety profiles similar or highly similar to those of the reference medicine. Alternatively, or in addition, the biosimilars described herein can be considered to have efficacy similar or highly similar to that of the reference medicine. As described herein, the biosimilars in Europe are compared with the reference medicines authorized by the EMA. However, in some cases, biosimilars may be compared in certain studies with biological medicines authorized outside the European Economic Area (non-EEA authorized "comparators").Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" is also relevant to biological medicinal products that have been or may be compared to comparators not authorized in the EEA. Certain biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications in the amino acid structure (including, for example, deletions, additions, and / or substitutions of amino acids), which do not significantly affect the function of the polypeptide. A biosimilar may contain an amino acid sequence having 97% or higher (e.g., 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of its reference medicinal product. A biosimilar may contain one or more post-translational modifications that are different from those of the reference medicinal product (e.g., but not limited to glycosylation, oxidation, deamidation, and / or truncation), provided that the differences do not result in a change in the safety and / or efficacy of the medicinal product, such modifications. A biosimilar may have the same or a different glycosylation pattern compared to the reference medicinal product. In particular, although not exclusive, a biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety issues associated with the reference medicinal product. Additionally, a biosimilar may differ from the reference medicinal product, for example, in its strength, pharmaceutical form, formulation, excipients, and / or presentation, provided that the safety and efficacy of the medicinal product are not affected. Compared to the reference medicinal product, a biosimilar may contain differences, for example, in pharmacokinetic (PK) and / or pharmacodynamic (PD) characteristics, but the biosimilar is still considered to be sufficiently similar to the reference medicinal product to obtain authorization or to be considered suitable for authorization. In some cases, compared to the reference medicinal product, a biosimilar exhibits different binding characteristics, where the different binding characteristics are not regarded by regulatory authorities such as the EMA as an obstacle to authorization as a similar biological medicinal product. The term "biosimilar" is also used synonymously by regulatory authorities in other countries and regions.

[0104] The term "chimeric antigen receptor" or alternatively "CAR" refers to a group of polypeptides, typically two polypeptides in the simplest embodiments, which, when in an immune effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and provide for intracellular signal generation. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), the cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule as defined below. In some aspects, the polypeptides of the group are contiguous with one another. In some embodiments, the group of polypeptides includes a dimerization switch that can couple the polypeptides to one another in the presence of a dimerizing molecule, e.g., can couple the antigen-binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is the ζ-chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from the co-stimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence located at the amino terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence located at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and CAR localization to the cell membrane.

[0105] II. Methods for Evaluating Proliferation Index

[0106] Embodiments of the present invention relate to methods for assessing the proliferation index of T cell populations. In some embodiments, the T cells are gene-edited T cells. As used herein, "gene-editing", "geneediting", and "genome editing" refer to a type of genetic modification in which DNA is permanently modified in the cell genome, e.g., inserting, deleting, modifying, or replacing DNA within the genome of the cell. In some embodiments, gene editing causes the expression of a DNA sequence to be silenced (sometimes referred to as gene knockout) or inhibited / reduced (sometimes referred to as gene knockdown). In some embodiments, gene editing causes the expression of a foreign protein (such as a chimeric antigen receptor (CAR)) in the cell.

[0107] In some embodiments, the method comprises:

[0108] (a) culturing a first portion of a gene-edited T cell population in a cell culture medium that does not have IL-2;

[0109] (b) adding an agent to the cell culture medium, wherein the agent is incorporated into the DNA of the cells during proliferation;

[0110] (c) measuring, at one or more time points, the amount of the agent incorporated into the gene-edited T cells; and

[0111] (d) calculating the proliferation index of the gene-edited T cell population based on the amount of the agent incorporated into the gene-edited T cells at the one or more time points.

[0112] In some embodiments, the gene-edited T cells are allowed to proliferate in a cell culture medium that does not have IL-2 for a period of time (e.g., 1 day, 7 days, 10 days, 14 days, etc.), and then the level of proliferation is evaluated after the proliferation period. Any of a variety of well-known methods can be used to evaluate the level of proliferation of the gene-edited T cells (and corresponding controls). In various examples, the method can use a fluorescence reader. Using a fluorescence reader easily allows for the detection of a fluorescence signal, which aids in evaluating the level of proliferation of the gene-edited T cells. For example, as further described below, features that are directly related to showing the level of proliferation can be detected.

[0113] According to the method of the present invention, the proliferation level of gene-edited T cells can be determined by any of a variety of known proliferation assays. These assays may fall into one of the following categories, which include assays involving: (i) measurement of DNA synthesis, (ii) detection of proliferation-specific cell markers, (iii) measurement of consecutive cell divisions by using cell membrane-binding dyes, (iv) measurement of cell DNA content, and (v) measurement of cell metabolism. For any of these methods, the proliferation level of gene-edited T cells in the absence of IL-2 is typically compared to the proliferation level of a positive control (such as a transformed cell line that grows independently of IL-2, e.g., Jurkat cells), as described below.

[0114] As described above, according to the method of the present invention, DNA synthesis assays can be used to determine the proliferation level of gene-edited T cells. In one example of such a method, the incorporation of a non-radioactive, modified nucleotide into the DNA of dividing cells is detected as a measure of proliferation. For example, 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analogue, can be used to assess active DNA synthesis. This analogue (e.g., EdU) can be added to the proliferating cells before the end of the above-mentioned proliferation period, e.g., about 24 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours before the end of the proliferation period. EdU can be added to the cell culture medium at a concentration of about 40 μM, about 20 μM, about 10 μM, about 5 μM, about 2.5 μM, about 1.25 μM, and / or about 0 μM. In some embodiments, EdU can be added to the cell culture medium at a concentration of about 20 μM.

[0115] The incorporated analogue (e.g., EdU) can be detected, for example, by a click reaction (copper-catalyzed azide-alkyne cycloaddition reaction), using, for example, a fluorescent probe (e.g., Amplex UltraRed reagent, EdU Alexa 488, etc.), thereby facilitating the detection of newly synthesized DNA by image-based techniques (see, e.g., Messele et al., Clin Diagn Lab Immunol. 7(4):687-92, 2000; Salic and Mitchison, Proc Natl Acad Sci USA 105(7):2415-20, 2008, the entire contents of which are hereby incorporated by reference in their entirety).

[0116] In some embodiments, cells are seeded in a 96-well plate on day 0. For each well, the number of TILs to be added can be 1e4, 1e5, 2e5, or 1e6. In some embodiments, 1e5 TILs are added to each well. For each well, the number of Jurkat cells to be added can be 1e3, 2e3, or 1e4. In some embodiments, 1e3 Jurkat cells are added to each well. In some embodiments, 2e3 Jurkat cells are added to each well. In some embodiments, each sample can be run in duplicate, triplicate, or quadruplicate.

[0117] EdU (20 μM) is added to the appropriate wells 24 hours before running the assay (e.g., on day 6 and / or day 9). The assay can be run on day 7 and / or day 10. To run the assay, 80 μl of medium is removed from each well, and 50 μl of EdU fixative is added to each well. The plate is incubated at room temperature for 5 minutes. The fixative is removed and the wells are washed with 200 μl of EdU wash buffer. Then 50 μl of EdU reaction mixture (containing deionized water, EdU master mix, EdU reaction additive, and HRP-azide) is added to each well and incubated at room temperature for 30 minutes. The reaction mixture is removed from the plate. Next, 200 μl of 1.5% BSA blocking solution is added to each well and incubated at room temperature in the dark for 5 minutes. The blocking solution is removed from the plate. Then the plate is washed 3 times with 200 μl of EdU wash buffer. The reaction is started by adding 100 μl of Amplex TM UltraRed reaction buffer, Amplex TM UltraRed reagent, and hydrogen peroxide solution Amplex TM UltraRed reaction mixture, and the plate is incubated at room temperature in the dark for 15 minutes. The reaction is terminated by adding 10 μl / well of Amplex TM UltraRed stop solution. Then the plate is read on a fluorescence microplate reader (recommended excitation wavelength is 568 nm and emission wavelength is 585 nm).

[0118] In other instances, 5-bromo-2'-deoxyuridine (BrdU) can be used to detect cell proliferation. When gene-edited T cells are cultured with a labeled medium containing BrdU, this pyrimidine analogue incorporates into newly synthesized DNA in place of thymidine. Detection of incorporated BrdU can be achieved using an anti-BrdU antibody (see, e.g., Porstmann et al., J Immunol Methods 82(1):169-79, 1985).

[0119] Another example for detecting DNA synthesis, as a measure of the proliferation of gene-edited T cells, makes use of radioactive nucleotides 3 H] thymidine ( 3 H] TdR), and involves detecting the incorporation of 3 H] TdR into the newly synthesized strands of chromosomal DNA during cell division (see, e.g., Denton, Methods Mol Biol 79:169-77, 1998). A scintillation counter can be used to measure the radioactivity in the DNA recovered from 3 H] TdR-treated cells.

[0120] As described above, in the context of the present invention, assays in which proliferation-specific cell markers are detected can also be utilized. An exemplary assay involves the detection of the nuclear-specific proliferation antigen Ki-67. Detection of Ki-67 protein expression in proliferating cells is achieved by using an anti-Ki-67 antibody, followed by imaging techniques or flow cytometry (see, e.g., Soares et al., J Immunol Methods 362(1-2):43-50, 2010). The Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in resting cells.

[0121] In other instances, cell membrane-binding dyes can be utilized in the context of the present invention to evaluate cell proliferation. Some exemplary dyes available include carboxyfluorescein succinimidyl ester (CFSE) and CellTrace TM Far Red. Such dyes cross the cytoplasmic membrane and covalently bind to all free amines on the cell surface and internally, and the dyes are retained for a relatively long period of time. The fluorescence signal after dye incorporation into cells can be detected by a fluorescence reader or a flow cytometer. Since the fluorescence gradually halves in daughter cells after each cell division, the detected signal can subsequently be used to monitor proliferation (see, e.g., Tario et al., J Vis Exp (70):e4287, 2012; and Filby et al., Methods 82:29-37, 2015).

[0122] Cell DNA content can also be measured to determine the extent of cell proliferation. An exemplary method that can be used is a cell proliferation assay that employs a green fluorescent nucleic acid dye and a background suppression dye that cannot penetrate living cells and inhibits the nucleic acid dye, thereby preventing staining of dead cells and cells with damaged cell membranes. Detection of the stained DNA is achieved by measuring fluorescence with a fluorescence reader (see, e.g., Jones et al., J Immunol Methods 254(1-2):85-98, 2001).

[0123] In other examples, cell metabolism can be evaluated in the context of the present invention to determine cell proliferation. Tetrazolium salts, such as MTT and MTS, can be used to evaluate cell metabolic activity, which reflects the number of living cells present in a sample. Under defined conditions, NAD(P)H-dependent cellular oxidoreductases are capable of reducing such tetrazolium salts to colored, insoluble formazan dyes. The amount of the colored product formed can be quantified by measuring the light absorption at a specific wavelength in the solution (see, e.g., Mosmann, J Immunol Methods 65(1-2):55-63, 1983; and Cory et al., Cancer Commun 3(7):207-12, 1991). Related compounds for measuring cell metabolic activity have a fluorescence-based readout that is proportional to the number of cells in a given sample (see, e.g., Ahmed et al., J Immunol Methods 170(2):211-24, 1994). Additionally, ATP bioluminescence can be used as a measure of cell proliferation, correlating the ATP concentration in a given sample with the number of living cells (see, e.g., Crouch et al., J Immunol Methods 160(1):81-8, 1993).

[0124] In the method according to the present invention, the proliferation level of gene-edited T cells in the absence of IL-2 can be represented numerically, such as the proliferation index (PI), which reflects the transition state of the gene-edited T cells. The proliferation level of gene-edited T cells in the absence of IL-2 can be compared with the proliferation level of a positive control sample of a cell line (e.g., Jurkat cells) that shows a transition to IL-2-independent growth, or with the proliferation level of a positive control sample of gene-edited T cells in the presence of IL-2, or with both. Additionally, the proliferation level of gene-edited T cells in the absence of IL-2 can be compared with the proliferation level of a negative control sample of non-gene-edited T cells (e.g., mock gene-edited T cells) in the absence of IL-2. Based on one or more of this information, the proliferation index (PI) according to one of the following expressions can be used as a measure of the transition state of the gene-edited T cells:

[0125] PI = (the proliferation level of gene-edited T cells in the absence of IL-2);

[0126] PI = (the proliferation level of gene-edited T cells in the absence of IL-2) - (the proliferation level of the negative control sample in the absence of IL-2);

[0127] PI = (the proliferation level of gene-edited T cells in the absence of IL-2) / (the proliferation level of the positive control sample in the absence of IL-2); or

[0128] PI = [(the proliferation level of gene-edited T cells in the absence of IL-2) - (the proliferation level of the negative control sample in the absence of IL-2)] / (the proliferation level of the positive control sample in the absence of IL-2).

[0129] In some embodiments, the gene-edited T cells are gene-edited TILs.

[0130] In some embodiments, the gene-edited T cells are CAR T cells, which are T cells comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain that binds to a tumor antigen as described herein (e.g., an antibody or antibody fragment, a TCR or TCR fragment), a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain as described herein) (e.g., an intracellular signaling domain comprising a co-stimulatory domain (e.g., a co-stimulatory domain as described herein) and / or a primary signaling domain (e.g., a primary signaling domain as described herein)). The CAR nucleic acid constructs, encoded proteins, vectors containing them, host cells, pharmaceutical compositions, and methods of administration and treatment related to the present invention are disclosed in detail in International Patent Application Publication No. WO2015142675, which is incorporated herein by reference in its entirety.

[0131] III. Gene-Edited TIL

[0132] Embodiments of the present invention relate to populations of gene-edited TILs, which are produced using a method comprising one or more steps of gene-editing at least a portion of the TILs to enhance the therapeutic effect of the TILs. As used herein, "gene-editing", "gene editing", and "genome editing" refer to a type of genetic modification in which DNA is permanently modified in the cell genome, e.g., inserted, deleted, modified, or replaced within the genome of the cell. In some embodiments, gene-editing causes the expression of a DNA sequence to be silenced (sometimes referred to as gene knockout) or inhibited / reduced (sometimes referred to as gene knockdown). According to embodiments of the present invention, gene-editing techniques are used to enhance the effectiveness of therapeutic TIL populations.

[0133] The method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, wherein the method further comprises gene-editing at least a portion of the TILs. According to additional embodiments, the method for expanding TILs into a therapeutic TIL population is carried out according to any embodiment of the methods described in WO 2018 / 081473 A1, WO 2018 / 129332 A1, or WO 2018 / 182817 A1, which patents are incorporated herein by reference in their entirety, wherein the method further comprises gene-editing at least a portion of the TILs. Thus, embodiments of the present invention provide a therapeutic TIL population that has been expanded according to any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, e.g., at least a portion of the therapeutic TIL population transferred to an infusion bag is permanently gene-edited.

[0134] In some embodiments, the gene-edited TIL population comprises reduced expression of a protein.

[0135] In some embodiments, the protein is PD-1.

[0136] In some embodiments, the protein is CTLA-4.

[0137] In some embodiments, the protein is LAG-3.

[0138] In some embodiments, the protein is CISH.

[0139] In some embodiments, the protein is TIGIT.

[0140] In some embodiments, the protein is CBL-B.

[0141] In some embodiments, the gene-edited TIL population comprises reduced expression of a first protein and a second protein.

[0142] In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein and the second protein are different.

[0143] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

[0144] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

[0145] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

[0146] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

[0147] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

[0148] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

[0149] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

[0150] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

[0151] In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

[0152] In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

[0153] In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

[0154] In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

[0155] In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

[0156] In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

[0157] In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

[0158] In some embodiments, the first protein is PD-1 and the second protein is CISH.

[0159] In some embodiments, the first protein is CISH and the second protein is PD-1.

[0160] In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

[0161] In some embodiments, the first protein is CBL-B and the second protein is PD-1.

[0162] In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

[0163] In some embodiments, the first protein is TIGIT and the second protein is PD-1.

[0164] In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

[0165] In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

[0166] In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

[0167] In some embodiments, the first protein is CISH and the second protein is CTLA-4.

[0168] In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

[0169] In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

[0170] In some embodiments, the first protein is LAG-3 and the second protein is CISH.

[0171] In some embodiments, the first protein is CISH and the second protein is LAG-3.

[0172] In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

[0173] In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

[0174] In some embodiments, the first protein is CISH and the second protein is CBL-B.

[0175] In some embodiments, the first protein is CBL-B and the second protein is CISH.

[0176] In some embodiments, the first protein or the second protein is PD-1.

[0177] In some embodiments, the first protein or the second protein is CTLA-4.

[0178] In some embodiments, the first protein or the second protein is LAG-3.

[0179] In some embodiments, the first protein or the second protein is CISH.

[0180] In some embodiments, the first protein or the second protein is CBL-B.

[0181] In some embodiments, the first protein or the second protein is TIGIT.

[0182] A.PD-1

[0183] One of the most studied targets for checkpoint blockade induction is the programmed death receptor (PD1 or PD-1, also known as PDCD1), which is a member of the T cell regulator CD28 superfamily. Its ligands, PD-L1 and PD-L2, are expressed in a variety of tumor cells, including melanoma. The interaction of PD-1 with PD-L1 inhibits T cell effector functions, leading to T cell exhaustion in the context of chronic stimulation and inducing T cell apoptosis in the tumor microenvironment. PD-1 may also play a role in tumor-specific immune surveillance escape.

[0184] According to certain embodiments, in the compositions and methods according to the present invention, the expression of PD-1 in TILs is silenced or reduced. For example, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the embodiments of the methods described herein, wherein the method includes gene editing at least a portion of the TILs by silencing or inhibiting the expression of PD-1. As described in more detail below, the gene editing process may involve the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at immune checkpoint genes such as PD-1. For example, the TALEN method can be used to silence or reduce the expression of PD-1 in TILs.

[0185] B.CTLA-4

[0186] CTLA-4 expression is induced upon T cell activation on activated T cells and competes with antigen-presenting cell-activated antigens CD80 and CD86 for binding. The interaction of CTLA-4 with CD80 or CD86 causes T cell inhibition and is used to maintain the balance of the immune response. However, inhibiting the interaction of CTLA-4 with CD80 or CD86 may prolong T cell activation and thus increase the level of the immune response to cancer antigens.

[0187] According to certain embodiments, in the compositions and methods according to the invention, the expression of CTLA-4 in TILs is silenced or reduced. According to certain embodiments, in the compositions and methods according to the invention, the expression of both PD-1 and CTLA-4 in TILs is silenced or reduced. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the embodiments of the methods described herein (e.g., the method shown in Process 2A or FIGS. 20 and 21), wherein the method includes gene editing at least a portion of the TILs by silencing or inhibiting the expression of CTLA-4. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at immune checkpoint genes such as CTLA-4. For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit the expression of CTLA-4 in TILs. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and CTLA-4 in TILs.

[0188] C.LAG-3

[0189] Lymphocyte activation gene 3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells following ligation of major histocompatibility complex (MHC) class II. Although its mechanism is not well understood, its modulation causes negative regulation of T cell function, thereby preventing tissue damage and autoimmunity. Thus, LAG-3 blockade may improve the anti-tumor response. See, e.g., Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

[0190] According to certain embodiments, in the compositions and methods according to the invention, the expression of LAG-3 in TILs is silenced or reduced. According to certain embodiments, in the compositions and methods according to the invention, the expression of both PD-1 and LAG-3 in TILs is silenced or reduced. For example, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the embodiments of the methods described herein (e.g., the method shown in Process 2A or FIGS. 20 and 21), wherein the method includes gene editing at least a portion of the TILs by silencing or inhibiting the expression of LAG-3. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at immune checkpoint genes such as LAG-3. According to certain embodiments, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit the expression of LAG-3 in TILs. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and LAG-3 in TILs.

[0191] D.Cish

[0192] Cish is a member of the suppressor of cytokine signaling (SOCS) family. Cish is induced by TCR stimulation in CD8+ T cells and inhibits the functional avidity of Cish for tumors. Gene deletion of Cish in CD8+ T cells may enhance the expansion, functional avidity, and cytokine multifunctionality of the CD8+ T cells, leading to a marked and sustained regression of established tumors. See, e.g., Palmer et al., Journal of Experimental Medicine, 212(12):2095 (2015).

[0193] According to certain embodiments, in the compositions and methods according to the invention, the expression of Cish in TILs is silenced or reduced. According to certain embodiments, in the compositions and methods according to the invention, the expression of both PD-1 and Cish in TILs is silenced or reduced. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein (e.g., the method shown in Process 2A or FIGS. 20 and 21), wherein the method includes gene editing at least a portion of the TILs by silencing or inhibiting the expression of Cish. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at immune checkpoint genes such as Cish. For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or inhibit the expression of Cish in TILs. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and Cish in TILs.

[0194] E.CBL-B

[0195] CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and is a negative regulator of T cell activation. Bachmaier et al., Nature, 2000, 403, 211–216; Wallner et al., Clin. Dev. Immunol. 2012, 692639.

[0196] According to certain embodiments, in the compositions and methods according to the invention, the expression of CBL-B in TILs is silenced or reduced. According to certain embodiments, in the compositions and methods according to the invention, the expression of both PD-1 and CBL-B in TILs is silenced or reduced. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein (e.g., the method shown in Process 2A or FIGS. 20 and 21), wherein the method includes gene editing at least a portion of the TILs by silencing or inhibiting the expression of CBL-B. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at immune checkpoint genes such as CBL-B. For example, the CRISPR method, the TALE method, or the zinc finger method can be used to silence or inhibit the expression of PKA in TILs. In some embodiments, TALEN knockout is used to silence CBL-B. In some embodiments, TALE-KRAB transcriptional repressor knock-in is used to silence CBL-B. More detailed information on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and CBL-B in TILs.

[0197] F. TIGIT

[0198] TIGIT is a cell surface protein expressed on regulatory T cells, memory T cells, and activated T cells. TIGIT belongs to the poliovirus receptor (PVR) immunoglobulin family and inhibits T cell activation. (Yu et al., Nat Immunol., 2009, 10(1):48-57).

[0199] According to certain embodiments, in the compositions and methods according to the present invention, the expression of TIGIT in TILs is silenced or reduced. According to certain embodiments, in the compositions and methods according to the present invention, the expression of both PD-1 and TIGIT in TILs is silenced or reduced. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein (e.g., the method shown in Process 2A or FIGS. 20 and 21), wherein the method includes gene editing at least a portion of the TILs by silencing or suppressing the expression of TIGIT. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at immune checkpoint genes such as TIGIT. For example, CRISPR methods, TALE methods, or zinc finger methods can be used to silence or suppress the expression of PKA in TILs. In some embodiments, TALEN knockouts are used to silence TIGIT. In some embodiments, TALE-KRAB transcriptional repressor knock-ins are used to silence TIGIT. More detailed information on these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some embodiments, the TALEN method can be used to silence or reduce the expression of PD-1 and TIGIT in TILs.

[0200] IV. Gene Editing Methods

[0201] As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that are genetically modified via gene editing to enhance their therapeutic effects. Embodiments of the present invention include gene editing by inserting nucleotides (RNA or DNA) into a TIL population to inhibit the expression of one or more proteins. Embodiments of the present invention also provide methods for expanding TILs into a therapeutic population, wherein the methods include gene editing of the TILs. There are several gene editing techniques available for genetically modifying TIL populations, which are suitable for use according to the present invention.

[0202] In some embodiments, methods of genetically modifying a TIL population include the step of stably incorporating a gene to produce one or more proteins. In one embodiment, methods of genetically modifying a TIL population include the step of retroviral transduction. In one embodiment, methods of genetically modifying a TIL population include the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine et al., Proc. Nat’l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In one embodiment, methods of genetically modifying a TIL population include the step of gammaretroviral transduction. Gammaretroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, methods of genetically modifying a TIL population include the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, transposase provided as mRNA (e.g., mRNA comprising a cap and poly-A tail). Suitable transposon-mediated gene transfer systems, including salmonid Tol2-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, are described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

[0203] In one embodiment, a method of genetically modifying a population of TILs includes the step of stably incorporating a gene to produce or inhibit (e.g., silence) one or more proteins. In one embodiment, a method of genetically modifying a population of TILs includes the step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297 - 306 and U.S. Patent Application Publication No. 2014 / 0227237A1, the disclosures of which and the patents are incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490, the disclosures of which patents are incorporated herein by reference. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method that includes the step of treating the TILs with a pulsed electric field to effect, manipulate, or cause defined and controlled, permanent or transient changes in the TILs, including the step of applying to the TILs a sequence of at least three individual, operator - controlled, independently programmed DC electric pulses having a field strength equal to or greater than 100 V / cm, wherein the sequence of at least three DC electric pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) the first pulse interval of a first set of two pulses of the at least three pulses is different from the second pulse interval of a second set of two pulses of the at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method that includes the step of treating the TILs with a pulsed electric field to effect, manipulate, or cause defined and controlled, permanent or transient changes in the TILs, including the step of applying to the TILs a sequence of at least three individual, operator - controlled, independently programmed DC electric pulses having a field strength equal to or greater than 100 V / cm, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In one embodiment, the electroporation method is a pulsed electroporation method that includes the step of treating the TILs with a pulsed electric field to effect, manipulate, or cause defined and controlled, permanent or transient changes in the TILs, including the step of applying to the TILs a sequence of at least three individual, operator - controlled, independently programmed DC electric pulses having a field strength equal to or greater than 100 V / cm, wherein at least two of the at least three pulses differ from each other in pulse width.In one embodiment, the electroporation method is a pulsed electroporation method, the pulsed electroporation method comprising the step of treating TILs with a pulsed electric field to effect, manipulate, or induce defined and controlled, permanent or transient changes in the TILs, including the step of applying to the TILs a sequence of at least three individual, operator-controlled, independently programmed DC electrical pulses having a field strength equal to or greater than 100 V / cm, wherein the first pulse interval of the first set of two pulses of the at least three pulses is different from the second pulse interval of the second set of two pulses of the at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method, the pulsed electroporation method comprising the step of treating TILs with a pulsed electric field to induce pore formation in the TILs, including the step of applying to the TILs a sequence of at least three DC electrical pulses having a field strength equal to or greater than 100 V / cm, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses are different from each other in pulse amplitude; (2) at least two of the at least three pulses are different from each other in pulse width; and (3) the first pulse interval of the first set of two pulses of the at least three pulses is different from the second pulse interval of the second set of two pulses of the at least three pulses, such that the induced pores are maintained for a relatively long period of time and such that the viability of the TILs is maintained. In one embodiment, the method of genetically modifying a population of TILs comprises the step of calcium phosphate transfection. The calcium phosphate transfection method (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) is known in the art and is described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayama, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a population of TILs comprises the step of liposome transfection.Liposomal transfection methods, such as those using a 1:1 (weight / weight) liposomal formulation of the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) in filtered water, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, as well as U.S. Patent Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes the step of transfecting using the methods described in U.S. Patent Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated herein by reference.

[0204] According to embodiments, the gene editing process may include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on the recognition of specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-stranded breaks at the target sequence. The DNA double-strand breaks then recruit endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homology-directed repair (HDR). Thus, repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, inhibit, or enhance) the target gene product.

[0205] The major classes of nucleases that have been developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR / Cas9). Based on the mode of DNA recognition, these nuclease systems can be broadly divided into two categories: ZFNs and TALENs achieve specific DNA binding via protein-DNA interactions, while CRISPR systems (such as Cas9) target specific DNA sequences through short RNA guide molecules that directly base pair with the target DNA and through protein-DNA interactions. See, e.g., Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

[0206] Non-limiting examples of gene editing methods that can be used with the TIL expansion method of the present invention include the CRISPR method, the TALE method, and the ZFN method, which are described in more detail below. According to an embodiment, the method for expanding TILs into a therapeutic population can be carried out according to any embodiment of the methods described herein (e.g., process 2A) or as described in WO 2018 / 081473A1, WO 2018 / 129332 A1, or WO 2018 / 182817 A1, wherein the method further comprises gene editing at least a portion of the TILs by one or more of the CRISPR method, the TALE method, or the ZFN method to generate TILs that can provide enhanced therapeutic effects. According to an embodiment, the improved therapeutic effects can be evaluated by comparing the gene-edited TILs with unmodified TILs in vitro, e.g., by evaluating in vitro effector functions, cytokine profiles, etc. compared to unmodified TILs.

[0207] In some embodiments of the present invention, electroporation is used to deliver gene editing systems, such as CRISPR, TALEN, and ZFN systems. In some embodiments of the present invention, the electroporation system is a flow electroporation system. An example of a suitable flow electroporation system for some embodiments of the present invention is the commercially available MaxCyte STX system. There are several alternative commercially available electroporation instruments that may be suitable for the present invention, such as the AgilePulse system or ECM 830 available from BTX-HarvardApparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza / Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the present invention, the electroporation system forms a closed sterile system with the remaining TIL expansion method. In some embodiments of the present invention, the electroporation system is a pulsed electroporation system as described herein and forms a closed sterile system with the remaining TIL expansion method.

[0208] A. TALE method

[0209] Methods for expanding TILs into a therapeutic population can be carried out according to any embodiment of the methods described herein or as described in WO2018 / 081473 A1, WO 2018 / 129332 A1 or WO 2018 / 182817A1, wherein the methods further comprise gene editing at least a portion of the TILs by the TALE method. According to a particular embodiment, use of the TALE method during the TIL expansion process causes the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population to be silenced or reduced. Alternatively, use of the TALE method during the TIL expansion process causes the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population to be enhanced.

[0210] TALE stands for "transcription activator-like effector" protein, which includes TALEN ("transcription activator-like effector nuclease"). The method of using the TALE system for gene editing may also be referred to herein as the TALE method. TALEs are naturally occurring proteins from the plant pathogen genus Xanthomonas and contain a DNA-binding domain consisting of a series of 33-35 amino acid repeat domains, each domain recognizing a single base pair. TALE specificity is determined by two hypervariable amino acids, which are called repeat variable diresidues (RVDs). Modular TALE repeats are joined together to recognize a contiguous DNA sequence. Specific RVDs in the DNA-binding domain recognize bases in the target locus, providing the structural features for assembling a predictable DNA-binding domain. The DNA-binding domain of the TALE is fused to the catalytic domain of the type IIS FokI endonuclease to form a targetable TALE nuclease. To induce site-specific mutations, two separate TALEN arms (separated by a spacer of 14-20 base pairs) bring the FokI monomers into close proximity for dimerization and generate a targeted double-strand break.

[0211] Several large-scale systematic studies using various assembly methods have shown that TALE repeats can be combined to recognize almost any user-defined sequence. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). The TALE methods and TALEN methods applicable to the present invention are described in U.S. Patent Application Publication Nos. US2011 / 0201118 A1; US 2013 / 0117869 A1; US2013 / 0315884 A1; US2015 / 0203871 A1 and US 2016 / 0120906 A1, the disclosures of which are incorporated herein by reference.

[0212] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TIL via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

[0213] The following table provides non-limiting examples of TALE nucleases targeting the PD-1 gene. In these examples, the targeted genomic sequence contains two 17-base pair (bp) long sequences (referred to as half-targets, shown in capital letters), which are separated by a 15-bp spacer (shown in lowercase letters). Each half-target is recognized by the repeat sequences of the half-TALE nucleases listed in the table. Thus, according to certain embodiments, the TALE nucleases of the present invention recognize and cleave target sequences selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:14. The TALEN sequences and gene editing methods are also described in Gautron et al., Molecular Therapy: Nucleic Acids, December 2017, Vol. 9: 312-321, which is incorporated herein by reference.

[0214] Table 3. TALEN PD-1 sequences.

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] Non-limiting examples of genes that can be enhanced by permanent gene editing of TIL via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

[0224] Examples of systems, methods, and compositions for altering the expression of a target gene sequence by the TALE method (and which can be used according to embodiments of the present invention) are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

[0225] B.2. Cas-CLOVER method

[0226] Methods for expanding TILs into a therapeutic population can be carried out according to any embodiment of the methods described herein (e.g., Procedure 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the methods further comprise gene editing at least a portion of the TILs by the Cas-CLOVER method. According to certain embodiments, use of the Cas-CLOVER method during the TIL expansion process results in silencing or reduction of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, use of the Cas-CLOVER method during the TIL expansion process results in enhancement of the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

[0227] Cas-CLOVER is a dimeric, high-fidelity site-specific nuclease (SSN) composed of a catalytically inactive SpCas9 (dCas9) fused to the nuclease domain of the type IIS restriction endonuclease from Clostridium Clo051 (Madison et al., “Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of T SCM-enriched allogeneic CAR-T cells,” Molecular Therapy-Nucleic Acids, 2022). This results in a nuclease whose activity depends on dimerization of the Clo051 nuclease domain, achieved by RNA-guided recognition of two adjacent 20-nt target sequences. Unlike paired nickase methods (e.g., when using the Cas9-D10A mutant), monomeric Cas-CLOVER does not introduce nicks or DSBs. Cas-CLOVER has been shown to have low off-target nuclease activity.

[0228] Exemplary Cas-CLOVER systems include those described in WO2019 / 126578, the contents of which are incorporated herein by reference in their entirety. In embodiments, the Cas-CLOVER system comprises a fusion protein that comprises, consists essentially of, or consists of a DNA targeting component and an effector molecule.

[0229] DNA targeting component

[0230] In embodiments, the DNA targeting component is capable of binding to a specific DNA sequence. In embodiments, the DNA targeting component is selected from, for example, DNA-binding oligonucleotides, DNA-binding proteins, DNA-binding protein complexes, and combinations thereof. One of ordinary skill in the art will recognize other suitable DNA-binding components.

[0231] In embodiments, the DNA targeting component comprises an oligonucleotide that targets a specific locus or loci in the genome. The oligonucleotide is selected from DNA, RNA, DNA / RNA hybrids, and combinations thereof.

[0232] In embodiments, the DNA targeting component comprises a nucleotide-binding protein or protein complex that binds to a binding oligonucleotide when bound to the target DNA. The protein or protein complex may be capable of recognizing a feature selected from an RNA-DNA heteroduplex, an R-loop, or a combination thereof. In embodiments, the DNA targeting component comprises a protein or protein complex that is capable of recognizing an R-loop, and the protein or protein complex is selected from Cas9, the Cascade complex, RecA, RNase H, RNA polymerase, DNA polymerase, or a combination thereof. In embodiments, the DNA targeting component comprises an engineered protein that is capable of binding to the target DNA. In embodiments, the DNA targeting component comprises a protein that is capable of binding to a DNA sequence selected from meganucleases, zinc finger arrays, transcription activator-like (TAL) arrays, and combinations thereof. In embodiments, the DNA targeting component comprises a protein containing a naturally occurring DNA-binding domain. In embodiments, the DNA targeting component comprises a bZIP domain, a helix-loop-helix, a helix-turn-helix, an HMG-box, a leucine zipper, a zinc finger, or a combination thereof. In embodiments, the DNA targeting component comprises an oligonucleotide that targets a specific locus in the genome. Exemplary oligonucleotides include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and any combination thereof. In embodiments, the DNA targeting component comprises a protein or protein complex that is capable of recognizing a feature selected from an RNA-DNA heteroduplex, an R-loop, and any combination thereof. Exemplary proteins or protein complexes that are capable of recognizing an R-loop include, but are not limited to, Cas9, the Cascade complex, RecA, RNase H, RNA polymerase, DNA polymerase, and any combination thereof. In embodiments, the protein or protein complex that is capable of recognizing an R-loop comprises Cas9. In embodiments, the DNA targeting component comprises a protein that is capable of binding to a DNA sequence selected from meganucleases, zinc finger arrays, TAL arrays, and any combination thereof. In embodiments, the DNA targeting component comprises an oligonucleotide that targets a target position in the genome and a protein that is capable of binding to the target DNA sequence.

[0233] In embodiments, the DNA targeting component comprises at least one guide RNA (gRNA), consists essentially of at least one guide RNA (gRNA), or consists of at least one guide RNA (gRNA). In embodiments, the DNA targeting component comprises two gRNAs, consists essentially of two gRNAs, or consists of two gRNAs, wherein the first gRNA specifically binds to the first strand of a double-stranded DNA target sequence and the second gRNA specifically binds to the second strand of the double-stranded DNA target sequence. Alternatively, in embodiments, the DNA targeting component comprises the DNA-binding domain of a transcription activator-like effector nuclease (TALEN, also referred to as a TAL protein), consists essentially of the DNA-binding domain, or consists of the DNA-binding domain. In embodiments, the DNA targeting component comprises the DNA-binding domain of a TALEN or TAL protein from the genus Xanthomonas or Ralstonia, consists essentially of the DNA-binding domain, or consists of the DNA-binding domain.

[0234] Effector molecule

[0235] In embodiments, the effector molecule is capable of generating a predetermined effect at a specific locus in the genome. Exemplary effector molecules include but are not limited to transcription factors (activators or repressors), chromatin remodeling factors, nucleases, exonucleases, endonucleases, transposases, methyltransferases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, helicases, fluorophores, or any combination thereof.

[0236] In embodiments, the effector molecule includes a transposase. In embodiments, the effector molecule includes a PB transposase (PBase). In embodiments, the effector molecule includes a nuclease. Non-limiting examples of nucleases include restriction endonucleases, homing endonucleases, S1 nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO endonuclease, or any combination thereof. In certain embodiments, the effector molecule includes a restriction endonuclease. In certain embodiments, the effector molecule includes a type IIS restriction endonuclease. In embodiments, the effector molecule includes an endonuclease. Non-limiting examples of endonucleases include AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, and Clo051. In embodiments, the effector molecule includes BmrI, BfiI, or Clo051.

[0237] In embodiments, the effector molecule includes a homodimer or heterodimer, consists essentially of a homodimer or heterodimer, or consists of a homodimer or heterodimer. In embodiments, the effector molecule includes a nuclease (optionally an endonuclease), consists essentially of a nuclease (optionally an endonuclease), or consists of a nuclease (optionally an endonuclease). In embodiments, the effector molecule (including those that include a homodimer or heterodimer) includes Cas9, a Cas9 nuclease domain, or a fragment thereof, consists essentially of Cas9, a Cas9 nuclease domain, or a fragment thereof, or consists of Cas9, a Cas9 nuclease domain, or a fragment thereof. In embodiments, Cas9 is catalytically inactive or “dead” Cas9 (SEQ ID NOs: 302 and 303 of WO2019 / 126578). In embodiments, Cas9 is a catalytically inactive or “dead” nuclease domain of Cas9. In embodiments, dCas9 is encoded by a shorter sequence derived from full-length, catalytically inactivated Cas9, herein referred to as “small” dCas9 or dSaCas9 (SEQ ID NO: 23 of WO2019 / 126578).

[0238] In embodiments of the fusion protein, the effector molecule comprises a homodimer or heterodimer of one or more type II nucleases, consists essentially of a homodimer or heterodimer of one or more type II nucleases, or consists of a homodimer or heterodimer of one or more type II nucleases. In embodiments of the fusion protein, the effector molecule comprises a homodimer or heterodimer of type II nucleases, consists essentially of a homodimer or heterodimer of type II nucleases, or consists of a homodimer or heterodimer of type II nucleases. In embodiments, the type II nucleases include one or more of AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I or Clo051.

[0239] In embodiments, effector molecules (including those that comprise homodimers or heterodimers) include Clo051, BfiI or BmrI, consist essentially of Clo051, BfiI or BmrI, or consist of Clo051, BfiI or BmrI. In embodiments, effector molecules (including those that comprise homodimers or heterodimers) include Cas9, the Cas9 nuclease domain or a fragment thereof that forms a heterodimer with Clo051, BfiI or BmrI, consist essentially of Cas9, the Cas9 nuclease domain or a fragment thereof that forms a heterodimer with Clo051, BfiI or BmrI, or consist of Cas9, the Cas9 nuclease domain or a fragment thereof that forms a heterodimer with Clo051, BfiI or BmrI. In embodiments, effector molecules (including those that comprise homodimers or heterodimers) include a catalytically inactive form of Cas9 (e.g., dCas9 or dSaCas9) or a fragment thereof that forms a heterodimer with Clo051, consist essentially of a catalytically inactive form of Cas9 (e.g., dCas9 or dSaCas9) or a fragment thereof that forms a heterodimer with Clo051, or consist of a catalytically inactive form of Cas9 (e.g., dCas9 or dSaCas9) or a fragment thereof that forms a heterodimer with Clo051. An exemplary Clo05l nuclease domain can comprise the following amino acid sequence, consist essentially of the following amino acid sequence, or consist of the following amino acid sequence: EGI KSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRH LGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVN PNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE KIRSGEMTIEELERAMFNNSEFILKY (SEQ ID NO:23).

[0240] In embodiments, effector molecules (including those comprising homodimers or heterodimers) include, consist essentially of, or consist of a DNA-binding domain of a TALEN or TAL protein from Xanthomonas or Ralstonia. In embodiments, effector molecules (including those comprising homodimers or heterodimers) include, consist essentially of, or consist of a DNA-binding domain of a TALEN or TAL protein from Xanthomonas or Ralstonia that forms a homodimer or heterodimer with Clo051, BfiI, or BmrI. In embodiments, effector molecules (including those comprising homodimers or heterodimers) include, consist essentially of, or consist of a DNA-binding domain of a TALEN or TAL protein from Xanthomonas or Ralstonia that forms a homodimer or heterodimer with Clo051.

[0241] Linkage

[0242] In embodiments, a fusion protein includes, consists essentially of, or consists of a DNA targeting component and an effector molecule. In embodiments, a nucleic acid sequence encoding one or more components of the fusion protein can be operably linked, for example, in an expression vector. In embodiments, the fusion protein is a chimeric protein. In embodiments, the fusion protein is encoded by one or more recombinant nucleic acid sequences. In embodiments, the fusion protein further includes a linker region operably linking the two components of the fusion protein. For example, in embodiments, the fusion protein includes, consists essentially of, or consists of a DNA targeting component and an effector molecule operably linked by a linker region. In embodiments, the DNA targeting component, linker region, and effector molecule can be encoded by one or more nucleic acid sequences inserted into an expression cassette and / or expression vector such that translation of the nucleic acid sequences produces the fusion protein. In embodiments, the fusion protein can include a non-covalent bond between the DNA targeting component and the effector molecule. The non-covalent bond can include an antibody, antibody fragment, antibody mimetic, or scaffold protein.

[0243] Fusion protein

[0244] In embodiments, the DNA targeting component comprises at least one gRNA, consists essentially of at least one gRNA, or consists of at least one gRNA, and the effector molecule comprises Cas9, a Cas9 nuclease domain or a fragment thereof, consists essentially of Cas9, a Cas9 nuclease domain or a fragment thereof, or consists of Cas9, a Cas9 nuclease domain or a fragment thereof. In embodiments, the DNA targeting component comprises at least one gRNA, consists essentially of at least one gRNA, or consists of at least one gRNA, and the effector molecule comprises inactivated Cas9 (dCas9) or an inactivated nuclease domain, consists essentially of inactivated Cas9 (dCas9) or an inactivated nuclease domain, or consists of inactivated Cas9 (dCas9) or an inactivated nuclease domain. In embodiments, the DNA targeting component comprises at least one gRNA, consists essentially of at least one gRNA, or consists of at least one gRNA, and the effector molecule comprises inactivated small Cas9 (dSaCas9), consists essentially of inactivated small Cas9 (dSaCas9), or consists of inactivated small Cas9 (dSaCas9). In embodiments, the effector molecule comprises Cas9, dCas9, dSaCas9 or a nuclease domain thereof and a second endonuclease, consists essentially of Cas9, dCas9, dSaCas9 or a nuclease domain thereof and a second endonuclease, or consists of Cas9, dCas9, dSaCas9 or a nuclease domain thereof and a second endonuclease. The second endonuclease may comprise a type IIS endonuclease, consist essentially of a type IIS endonuclease, or consist of a type IIS endonuclease, including, but not limited to, one or more of the following: AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051.

[0245] In embodiments of the fusion protein, the DNA targeting component comprises, consists essentially of, or consists of the DNA binding domain of a transcription activator-like effector nuclease (TALEN, also referred to as a TAL protein), and the effector molecule comprises, consists essentially of, or consists of an endonuclease. In embodiments of the fusion proteins of the present disclosure, the DNA targeting component comprises, consists essentially of, or consists of the DNA binding domain of a TALEN or TAL protein from the genus Xanthomonas or Ralstonia, and the effector molecule comprises, consists essentially of, or consists of a type IIS endonuclease, including but not limited to one or more of the following: AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I or Clo051.

[0246] In certain embodiments, an exemplary dCas9-Clo051 fusion protein can comprise the amino acid sequence of SEQ ID NO: 305 or 307 of WO2019 / 126578 or the nucleic acid sequence of SEQ ID NO: 306 or 308 of WO2019 / 126578, consist essentially of, or consist of the amino acid sequence or nucleic acid sequence.

[0247] Construct

[0248] In embodiments, the nuclease domain includes dCas9 and Clo051, consists essentially of dCas9 and Clo051, or consists of dCas9 and Clo051. In embodiments, the nuclease domain includes dSaCas9 and Clo051, consists essentially of dCas9 and Clo051, or consists of dCas9 and Clo051. In embodiments, the nuclease domain includes Xanthomonas - TALE and Clo051, consists essentially of Xanthomonas - TALE and Clo051, or consists of Xanthomonas - TALE and Clo051. In embodiments, the nuclease domain includes Ralstonia - TALE and Clo051, consists essentially of Ralstonia - TALE and Clo051, or consists of Ralstonia - TALE and Clo051. In embodiments, the fusion protein includes dCas9 - Clo051, dSaCas9 - Clo051, Xanthomonas - TALE - Clo051 or Ralstonia - TALE - Clo051. In embodiments, the vector encoding the fusion protein includes Csy4 - T2A - Clo051 - G4S linker - dCas9 (Streptoccocus pyogenes) or pRT1 - Clo051 - dCas9 dual NLS.

[0249] According to some embodiments, the Cas - CLOVER system comprises a fusion protein that includes a DNA targeting component and an effector molecule, wherein the DNA targeting component hybridizes to a target sequence of a DNA molecule in a TIL, wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule, and the effector molecule cleaves the DNA molecule, thereby altering the expression of at least one immune checkpoint molecule.

[0250] According to certain embodiments, the Cas-CLOVER method includes silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas-CLOVER system (e.g., dCas9-Clo051, dSaCas9-Clo051, Xanthomonas-TALE-Clo051, or Ralstonia-TALE-Clo051 fusion protein) that is specific for the target DNA sequence of an immune checkpoint gene. The fusion protein can be delivered as DNA, mRNA, or protein. When the genome comes into contact with the Cas-CLOVER system, one or more strands of the target double-stranded DNA can be cleaved. If the cleavage is carried out in the presence of one or more DNA repair pathways or their components, the Cas-CLOVER method will disrupt gene expression or modify the genomic sequence by insertion, deletion, or substitution of one or more base pairs. DSBs can be repaired in cells by non-homologous end joining (NHEJ), a mechanism that often causes insertions or deletions (indels) in the DNA. Indels typically result in a frameshift, causing the loss of a functional allele; for example, by introducing a premature stop codon within the open reading frame (ORF) of the target gene. According to certain embodiments, the result is the loss of a functional mutation within the target immune checkpoint gene.

[0251] Alternatively, DSBs induced by the Cas-CLOVER system can be repaired by homology-directed repair (HDR) rather than NHEJ. While NHEJ-mediated DSB repair typically disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide variations to large insertions. According to some embodiments, HDR is used for gene editing of immune checkpoint genes by delivering a DNA repair template containing the desired sequence into TILs with the Cas-CLOVER system. The repair template preferably contains the desired edit as well as additional homologous sequences immediately upstream and downstream of the target gene (commonly referred to as the left and right homology arms).

[0252] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TIL via the Cas-CLOVER method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.

[0253] Examples of systems, methods, and compositions for altering the expression of a target gene sequence by the Cas-CLOVER method (and which can be used according to embodiments of the present invention) are described in WO2019126578, US2017 / 0107541, US2017 / 0114149, US2018 / 0187185, and U.S. Patent No. 10,415,024, the contents of which are incorporated herein by reference in their entirety. Resources for performing the Cas-CLOVER method, such as CLOVER mRNA and Cas-CLOVER mRNA constructs, are commercially available from companies such as Demeetra and Hera Biolabs.

[0254] According to some embodiments, a method for expanding tumor-infiltrating lymphocytes (TIL) into a therapeutic TIL population includes:

[0255] (a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

[0256] (b) adding the tumor fragments to a closed system;

[0257] (c) performing a first expansion to produce a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing OKT-3 and / or a 4-1BB agonist antibody for about 3 to 11 days, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area;

[0258] (d) Stimulate the second TIL population by adding OKT-3 and culture for about 1 day to 3 days, wherein the transition from step (c) to step (d) can occur without opening the system;

[0259] (e) Perform aseptic electroporation on the second TIL population to effect transfer of at least one gene editor into a plurality of cells in the second TIL population;

[0260] (f) Allow the second TIL population cells to stand for about 1 day;

[0261] (g) Perform a second expansion to generate a third TIL population by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs), wherein the second expansion is performed for about 7 days to 11 days to obtain the third TIL population, 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) can occur without opening the system;

[0262] (h) Harvest the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) can occur without opening the system, wherein the harvested TIL population is a therapeutic TIL population;

[0263] (i) Transfer the harvested TIL population to an infusion bag, wherein the transfer from step (h) to (i) can occur without opening the system; and

[0264] (j) Optionally cryopreserve the harvested TIL population using a cryopreservation medium,

[0265] wherein the electroporation step comprises delivering at least one gene editor system comprising a Cas-CLOVER system, wherein at least one gene editor system modulates the expression of at least one checkpoint protein in a plurality of cells of the second TIL population.

[0266] According to some embodiments, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises:

[0267] (a) Obtain a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

[0268] (b) Add the tumor fragments to a closed system;

[0269] (c) Performing a first expansion to generate a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing OKT-3 and / or 4-1BB agonist antibody for about 3 to 11 days, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area;

[0270] (d) Stimulating the second TIL population by adding OKT-3 and culturing for about 1 to 3 days to obtain a second TIL population, wherein the transition from step (c) to step (d) can occur without opening the system;

[0271] (e) Performing a sterile electroporation on the second TIL population to effect the transfer of at least one gene editor to a plurality of cells in the second TIL population;

[0272] (f) Allowing the second TIL population cells to stand for about 1 day;

[0273] (g) Performing a second expansion to generate a third TIL population by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen-presenting cells (APCs), wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, 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) can occur without opening the system;

[0274] (h) Harvesting the therapeutic TIL population obtained in step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) can occur without opening the system, wherein the harvested TIL population is a therapeutic TIL population;

[0275] (i) Transferring the harvested TIL population to an infusion bag, wherein the transfer from step (h) to (i) can occur without opening the system; and

[0276] (j) Optionally cryopreserving the harvested TIL population using a cryopreservation medium,

[0277] wherein the electroporation step comprises delivering at least one gene editor system, the gene editor system comprising a Cas-CLOVER system, and the at least one gene editor system regulates the expression of at least one checkpoint protein in a plurality of cells of the second TIL population.

[0278] V. Method of treating cancer patients using gene-edited TIL

[0279] Some embodiments disclosed herein provide a method for treating a cancer patient, the method comprising:

[0280] (a) Generating a gene-edited TIL population from a tumor excised from a cancer patient;

[0281] (b) Evaluating the proliferation index of the gene-edited TIL population using the methods disclosed herein; and

[0282] (c) Administering a therapeutically effective dose of the gene-edited TILs to the cancer patient if the proliferation index of the gene-edited TIL population is lower than the proliferation index of a reference value.

[0283] According to the method of the present invention, the proliferation level of gene-edited T cells in the absence of IL-2 can be represented numerically, such as a proliferation index (PI), which reflects the transition state of the gene-edited T cells. The proliferation level of gene-edited T cells in the absence of IL-2 can be compared with the proliferation level of a positive control sample of a cell line (e.g., Jurkat cells) that shows a transition to IL-2-independent growth, or with the proliferation level of a positive control sample of gene-edited T cells in the presence of IL-2, or with both. In addition, the proliferation level of gene-edited T cells in the absence of IL-2 can be compared with the proliferation level of a negative control sample of non-gene-edited T cells (e.g., mock gene-edited T cells) in the absence of IL-2. Based on one or more of this information, the proliferation index (PI) according to the following expression can be used as a measure of the transition state of gene-edited T cells:

[0284] PI = (the proliferation level of gene-edited T cells in the absence of IL-2);

[0285] PI = (the proliferation level of gene-edited T cells in the absence of IL-2) - (the proliferation level of the negative control sample in the absence of IL-2);

[0286] PI = (the proliferation level of gene-edited T cells in the absence of IL-2) / (the proliferation level of the positive control sample in the absence of IL-2); or

[0287] PI = [(the proliferation level of gene-edited T cells in the absence of IL-2) - (the proliferation level of the negative control sample in the absence of IL-2)] / (the proliferation level of the positive control sample in the absence of IL-2).

[0288] In some embodiments, the reference value can be the proliferation index of a positive control. In some embodiments, the reference value can be 90% of the proliferation index of a positive control. In some embodiments, the reference value can be 80% of the proliferation index of a positive control. In some embodiments, the reference value can be 70% of the proliferation index of a positive control. In some embodiments, the reference value can be 60% of the proliferation index of a positive control. In some embodiments, the reference value can be 50% of the proliferation index of a positive control. In some embodiments, the reference value can be 40% of the proliferation index of a positive control. In some embodiments, the reference value can be 30% of the proliferation index of a positive control. In some embodiments, the reference value can be 20% of the proliferation index of a positive control. In some embodiments, the reference value can be 10% of the proliferation index of a positive control. In some embodiments, the reference value can be 5% of the proliferation index of a positive control.

[0289] In some embodiments, the reference value can be the proliferation index of a negative control. In some embodiments, the reference value can be the proliferation index of a negative control + / - 2 StdDev.

[0290] In some embodiments, the reference value can be from about 0 to about 0.9. In some embodiments, the reference value can be from about 0 to about 0.8. In some embodiments, the reference value can be from about 0 to about 0.7. In some embodiments, the reference value can be from about 0 to about 0.9. In some embodiments, the reference value can be from about 0 to about 0.6. In some embodiments, the reference value can be from about 0 to about 0.5. In some embodiments, the reference value can be from about 0 to about 0.4. In some embodiments, the reference value can be from about 0 to about 0.3. In some embodiments, the reference value can be from about 0 to about 0.2. In some embodiments, the reference value can be from about 0 to about 0.1. In some embodiments, the reference value can be from about 0 to about 0.05. In some embodiments, the reference value can be about 0.9. In some embodiments, the reference value can be about 0.9. In some embodiments, the reference value can be about 0.8. In some embodiments, the reference value can be about 0.7. In some embodiments, the reference value can be about 0.6. In some embodiments, the reference value can be about 0.5. In some embodiments, the reference value can be about 0.4. In some embodiments, the reference value can be about 0.3. In some embodiments, the reference value can be about 0.2. In some embodiments, the reference value can be about 0.1. In some embodiments, the reference value can be about 0.05.

[0291] The compositions and methods described herein can be used in methods of treating diseases. In some embodiments, they are used to treat hyperproliferative disorders, such as cancer, in adult or pediatric patients. They can also be used to treat other disorders as described herein and in the following paragraphs.

[0292] In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of: anal cancer, bladder cancer, breast cancer (including triple negative breast cancer), bone cancer, cancer caused by human papillomavirus (HPV), central nervous system related cancers (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectodermal tumors), cervical cancer (including cervical squamous cell carcinoma, cervical adenosquamous carcinoma, 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), hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, and pharyngeal cancer), kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC), metastatic NSCLC, and small cell lung cancer), melanoma (including uveal melanoma, choroidal melanoma, ciliary body melanoma, iris melanoma, or metastatic melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, kidney 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.

[0293] In some embodiments, the hyperproliferative disorder is hematological malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of: chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the cancer is a hematological malignancy. In some embodiments, the present invention includes a method of treating a patient having cancer using TIL, MIL, or PBL modified to downregulate one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-B, wherein the cancer is a hematological malignancy. In some embodiments, the present invention includes a method of treating a patient having cancer using MIL or PBL modified to downregulate one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-BR, wherein the cancer is a hematological malignancy.

[0294] In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, which recur or are difficult to treat with at least one prior therapy, including chemotherapy, radiotherapy, or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers, which recur or are difficult to treat with at least two prior therapies, including chemotherapy, radiotherapy, and / or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers, which recur or are refractory to treatment with at least three prior therapies, including chemotherapy, radiotherapy, and / or immunotherapy.

[0295] In some embodiments, the cancer is a microsatellite instability-high (MSI-H) cancer or a mismatch repair deficient (dMMR) cancer. Thus, MSI-H and dMMR cancers and assays have been described in Kawakami et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosure of which is incorporated herein by reference.

[0296] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the cancer is difficult to treat with a BRAF inhibitor and / or a MEK inhibitor. In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the cancer is difficult to treat with a BRAF inhibitor selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the cancer is difficult to treat with a MEK inhibitor selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the cancer is difficult to treat with a BRAF inhibitor and a MEK inhibitor, the BRAF inhibitor being selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof, and the MEK inhibitor being selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof.

[0297] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the cancer is a pediatric cancer.

[0298] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the cancer is uveal melanoma.

[0299] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the uveal melanoma is choroidal melanoma, ciliary body melanoma or iris melanoma.

[0300] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the pediatric cancer is neuroblastoma.

[0301] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the pediatric cancer is sarcoma.

[0302] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the sarcoma is osteosarcoma.

[0303] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the sarcoma is soft tissue sarcoma.

[0304] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma or primitive neuroectodermal tumor (PNET).

[0305] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the pediatric cancer is a central nervous system (CNS)-related cancer. In some embodiments, the pediatric cancer is difficult to treat with chemotherapy. In some embodiments, the pediatric cancer is difficult to treat with radiotherapy. In some embodiments, the pediatric cancer is difficult to treat with dinutuximab.

[0306] In some embodiments, the present invention includes a method of treating a patient having cancer, wherein the CNS-related cancer is medulloblastoma, pineoblastoma, glioma, ependymoma or glioblastoma.

[0307] The compositions and methods described herein can be used in a method for treating cancer that is difficult to treat or is resistant to prior anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient is a primary refractory patient to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has not shown a prior response to anti-PD-1 or anti-PD-L1 antibodies. In some embodiments, the patient has shown a prior response to anti-PD-1 or anti-PD-L1 antibodies, and subsequently the patient's cancer has progressed. In some embodiments, the cancer is difficult to treat with a combination of an anti-CTLA-4 antibody and / or an anti-PD-1 or anti-PD-L1 antibody and 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 is a platinum doublet chemotherapeutic agent. In some embodiments, the platinum doublet therapy comprises a first chemotherapeutic agent and a second chemotherapeutic agent, and the first chemotherapeutic agent is selected from the group consisting of cisplatin and carboplatin, and the second chemotherapeutic agent is selected from the group consisting of vinorelbine, gemcitabine, and taxane (including, for example, paclitaxel, docetaxel, or nab-paclitaxel). In some embodiments, the platinum doublet chemotherapeutic agent is combined with pemetrexed.

[0308] In some embodiments, the NSCLC is PD-L1 negative and / or from a patient with cancer expressing PD-L1 and a tumor proportion score (TPS) < 1%, as described elsewhere herein.

[0309] In some embodiments, the NSCLC is difficult to treat with a combination therapy comprising an anti-PD-1 or anti-PD-L1 antibody and a platinum doublet therapy, wherein the platinum doublet therapy comprises:

[0310] i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin,

[0311] ii) a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxane (including, for example, paclitaxel, docetaxel, or nab-paclitaxel).

[0312] In some embodiments, the NSCLC is difficult to treat with a combination therapy comprising an anti-PD-1 or anti-PD-L1 antibody, pemetrexed, and a platinum doublet therapy, wherein the platinum doublet therapy comprises:

[0313] i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin,

[0314] ii) a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel, docetaxel, or albumin-bound paclitaxel).

[0315] In some embodiments, NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient has not been treated. In some embodiments, NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, NSCLC has been previously treated with a chemotherapeutic agent. In some embodiments, NSCLC has been previously treated with a chemotherapeutic agent but is no longer being treated with a chemotherapeutic agent. In some embodiments, the NSCLC patient has not been treated with PD-1 / PD-L1. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the NSCLC patient has NSCLC that has not been treated or has received chemotherapy but has not been treated with anti-PD-1 / PD-L1. In some embodiments, the NSCLC patient has not been treated or has been treated after chemotherapy but has not been treated with anti-PD-1 / PD-L1 and has low PD-L1 expression. In some embodiments, the NSCLC patient has a large mass at baseline. In some embodiments, the subject has a large mass at baseline and has low PD-L1 expression. In some embodiments, the NSCLC patient does not have detectable PD-L1 expression. In some embodiments, the NSCLC patient has not been treated or has been treated after chemotherapy but has not been treated with anti-PD-1 / PD-L1 and does not have detectable PD-L1 expression. In some embodiments, the patient has a large mass at baseline and does not have detectable PD-L1 expression. In some embodiments, the NSCLC patient has NSCLC that has not been treated or has undergone chemotherapy (e.g., after a chemotherapeutic agent), but has not been treated with anti-PD-1 / PD-L1, and the patient has low PD-L1 expression and / or has a large mass at baseline. In some embodiments, a large mass is indicated when the maximum tumor diameter measured in the transverse or coronal plane is greater than 7 cm. In some embodiments, a large mass is indicated when the short axis diameter of the enlarged lymph node is 20 mm or longer. In some embodiments, the chemotherapeutic agent includes the standard of care treatment for NSCLC.

[0316] In some embodiments, PD-L1 expression is determined by tumor proportion score. In some embodiments, a subject with a refractory NSCLC tumor has a tumor proportion score (TPS) of <1%. In some embodiments, a subject with a refractory NSCLC tumor has a TPS of ≥1%. In some embodiments, a subject with refractory NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody, and the tumor proportion score was determined prior to said anti-PD-1 and / or anti-PD-L1 antibody treatment. In some embodiments, a subject with refractory NSCLC has been previously treated with an anti-PD-L1 antibody, and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment.

[0317] In some embodiments, PD-L1 expression is determined by tumor proportion score using one or more of the testing methods described herein. In some embodiments, a subject or patient with an NSCLC tumor has a tumor proportion score (TPS) of <1%. In some embodiments, the NSCLC tumor has a TPS of ≥1%. In some embodiments, a subject or patient with NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody, and the tumor proportion score was determined prior to said anti-PD-1 and / or anti-PD-L1 antibody treatment. In some embodiments, a subject or patient with NSCLC has been previously treated with an anti-PD-L1 antibody, and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment. In some embodiments, a subject or patient with a refractory or resistant NSCLC tumor has a tumor proportion score (TPS) of <1%. In some embodiments, a subject or patient with a refractory or resistant NSCLC tumor has a TPS of ≥1%. In some embodiments, a subject or patient with refractory or resistant NSCLC has been previously treated with an anti-PD-1 and / or anti-PD-L1 antibody, and the tumor proportion score was determined prior to said anti-PD-1 and / or anti-PD-L1 antibody treatment. In some embodiments, a subject or patient with refractory or resistant NSCLC has been previously treated with an anti-PD-L1 antibody, and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment. In some embodiments, NSCLC is an NSCLC that exhibits a tumor proportion score (TPS), or percentage of viable tumor cells collected from a patient prior to anti-PD-1 or anti-PD-L1 therapy, that shows partial or complete membranous staining at any intensity for PD-L1 protein of less than 1% (TPS < 1%). In some embodiments, NSCLC is an NSCLC that exhibits a TPS selected from the group consisting of: 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 the group consisting of: 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%. The TPS can be measured by methods known in the art, such as those described in Hirsch et al. J. Thorac. Oncol. 2017, 12, 208 - 222 or those used to determine the TPS prior to treatment with pembrolizumab or other anti - PD - 1 or anti - PD - L1 therapies. Methods approved by the U.S. Food and Drug Administration for measuring the TPS can also be used. In some embodiments, the PD - L1 is exosomal PD - L1. In some embodiments, the PD - L1 is present in circulating tumor cells.

[0318] In some embodiments, 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, complete membrane staining includes approximately 100% of the membrane staining.

[0319] In some embodiments, testing for PD-L1 can involve measuring the level of PD-L1 in a patient's serum. In these embodiments, measuring PD-L1 in the patient's serum removes the uncertainties of tumor heterogeneity and patient discomfort from serial biopsies.

[0320] In some embodiments, elevated soluble PD-L1 is associated with a worse prognosis in NSCLC compared to baseline or standard levels. See, e.g., Okuma et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli et al., Oncotarget, 2018, 9, 17554–17563. In some embodiments, PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is expressed on circulating tumor cells.

[0321] In some embodiments, a subject or patient with non-small cell lung cancer (NSCLC) is characterized by at least one of the following:

[0322] i. A predetermined tumor proportion score (TPS) of PD-L1 < 1%,

[0323] ii. A TPS score of PD-L1 of 1% - 49%, or

[0324] iii. A predetermined absence of one or more driver mutations,

[0325] wherein the driver mutations are selected from the group consisting of: EGFR mutations, EGFR insertions, EGFR exon 20 mutations, KRAS mutations, BRAF mutations, ALK mutations, c-ROS mutations (ROS1 mutations), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and / or altered MET signaling, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations, and GNA11 mutations.

[0326] In other embodiments, the present invention provides a method for treating a subject with cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population described herein.

[0327] In other embodiments, the present invention provides a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition described herein.

[0328] In other embodiments, the present invention provides a method for treating a subject having cancer described herein, the method being modified such that a non-myeloablative lymphodepletion regimen has been administered to the subject prior to separately administering a therapeutically effective dose of the therapeutic TIL population and the TIL composition described herein.

[0329] In other embodiments, the present invention provides a method for treating a subject having cancer described herein, the method being modified such that the non-myeloablative lymphodepletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m2 / day for five days.

[0330] In other embodiments, the present invention provides a method for treating a subject having cancer described herein, the method being modified to further comprise the step of treating the subject with a high-dose IL-2 regimen starting on the second day after administering the TIL cells to the subject.

[0331] In other embodiments, the present invention provides a method for treating a subject having cancer described herein, the method being modified such that the high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU / kg by intravenous infusion as a 15-minute bolus every eight hours until tolerance.

[0332] In other embodiments, the present invention provides a method for treating a subject having cancer described herein, the method being modified such that the cancer is a solid tumor.

[0333] In other embodiments, the present invention provides a method for treating a subject having cancer described herein, the method being modified such that the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer or renal cell carcinoma.

[0334] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0335] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is melanoma.

[0336] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is metastatic melanoma.

[0337] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is HNSCC.

[0338] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is cervical cancer.

[0339] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is NSCLC.

[0340] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is metastatic NSCLC.

[0341] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is glioblastoma (including GBM).

[0342] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is gastrointestinal cancer.

[0343] In other embodiments, the present invention provides methods for a subject having a cancer as described herein, the methods being modified such that the cancer is a highly mutated cancer.

[0344] In other embodiments, the present invention provides methods for treating a subject having a cancer as described herein, the methods being modified such that the cancer is a pediatric highly mutated cancer.

[0345] In other embodiments, the present invention provides a therapeutic TIL population as described herein for use in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

[0346] In other embodiments, the present invention provides a TIL composition as described herein for use in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

[0347] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which has been modified such that a non-myeloablative lymphodepletion regimen has been administered to the subject prior to administering to the subject a therapeutically effective dose of the therapeutic TIL population or TIL composition as described herein.

[0348] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which has been modified such that the non-myeloablative lymphodepletion regimen comprises the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for five days.

[0349] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which has been modified to further comprise the step of treating the patient with a high-dose IL-2 regimen starting on the second day after administering the TIL cells to the patient.

[0350] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which has been modified such that the high-dose IL-2 regimen comprises administering 600,000 or 720,000 IU / kg by intravenous infusion as a 15-minute bolus every eight hours until tolerance.

[0351] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which has been modified such that the cancer is a solid tumor.

[0352] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer or renal cell carcinoma.

[0353] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0354] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is melanoma.

[0355] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is metastatic melanoma.

[0356] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is HNSCC.

[0357] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is cervical cancer.

[0358] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is NSCLC.

[0359] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is metastatic NSCLC.

[0360] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is glioblastoma.

[0361] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is gastrointestinal cancer.

[0362] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is a highly mutated cancer.

[0363] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition as described herein, which is modified such that the cancer is a pediatric highly mutated cancer.

[0364] In other embodiments, the present invention provides the use of a therapeutic TIL population as described herein in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

[0365] In other embodiments, the present invention provides the use of the TIL composition described in any of the preceding paragraphs in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

[0366] In other embodiments, the present invention provides the use of a therapeutic TIL population as described herein or a TIL composition as described herein in a method of treating cancer in a patient, the method comprising administering to the patient a non-myeloablative lymphodepleting regimen and then administering to the subject a therapeutically effective dose of the therapeutic TIL population described in any of the preceding paragraphs or a therapeutically effective dose of the TIL composition as described herein.

[0367] 1. Lymphodepleting pretreatment of the patient

[0368] In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TIL according to the present disclosure. In some embodiments, the present invention includes a TIL population for treating cancer in a patient who has been pre-treated with non-myeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide at 60 mg / kg / d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine at 25 mg / m 2 / d for 5 days (from day 27 to day 23 before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion (on day 0) according to the present disclosure, the patient receives an intravenous infusion of 720,000 IU / kg of IL-2 (aldesleukin, commercially available as PROLEUKIN) intravenously every 8 hours until physiological tolerance. In certain embodiments, the TIL population is used for the combined treatment of cancer with IL-2, where IL-2 is administered after the TIL population.

[0369] The experimental results indicate that lymphodepletion prior to the adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing the therapeutic effect by eliminating regulatory T cells and competing elements of the immune system ('cytokine sink'). Accordingly, some embodiments of the present invention utilize a lymphocyte depletion step (sometimes also referred to as 'immunosuppressive conditioning') in the patient prior to the introduction of the TILs of the present invention.

[0370] Generally, lymphodepletion is achieved using the administration of fludarabine or cyclophosphamide (the active form is known 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 herein by reference in their entirety.

[0371] In some embodiments, fludarabine is administered at a concentration of 0.5 μg / mL to 10 μg / mL. In some embodiments, fludarabine is administered at a concentration of 1 μg / mL. In some embodiments, fludarabine is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or longer. In some embodiments, fludarabine is administered at a dose of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day. In some embodiments, fludarabine is administered at 35 mg / kg / day for 2 days - 7 days. In some embodiments, fludarabine is administered at 35 mg / kg / day for 4 days - 5 days. In some embodiments, fludarabine is administered at 25 mg / kg / day for 4 days - 5 days.

[0372] In some embodiments, the active form of cyclophosphamide, mafosfamide, is obtained by administering cyclophosphamide at a concentration of 0.5 μg / mL to 10 μg / mL. In some embodiments, the active form of cyclophosphamide, mafosfamide, is obtained by administering cyclophosphamide at a concentration of 1 μg / mL. In some embodiments, cyclophosphamide is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or longer. In some embodiments, cyclophosphamide is administered at a dose of 100 mg / m 2 / day, 150 mg / m 2 / day, 175 mg / m 2 / day, 200 mg / m 2 / day, 225 mg / m 2 / day, 250 mg / m 2 / day, 275 mg / m 2 / day, or 300 mg / m 2 / day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.v.). In some embodiments, cyclophosphamide is administered at a dose of 35 mg / kg / day for 2 to 7 days. In some embodiments, cyclophosphamide is administered intravenously at a dose of 250 mg / m 2 / day for 4 to 5 days. In some embodiments, cyclophosphamide is administered intravenously at a dose of 250 mg / m 2 / day for 4 days.

[0373] In some embodiments, lymphodepletion is performed by co-administering fludarabine and cyclophosphamide to a patient. In some embodiments, fludarabine is administered intravenously at a dose of 25 mg / m 2 / day and cyclophosphamide is administered intravenously at a dose of 250 mg / m 2 / day for 4 days.

[0374] In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for five days.

[0375] In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg / m 2 / day for two days and fludarabine at a dose of 25 mg / m 2 / day for five days, wherein both cyclophosphamide and fludarabine are administered for the first two days and wherein lymphodepletion is performed for a total of five days.

[0376] In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of about 50 mg / m 2 / day for two days and fludarabine at a dose of about 25 mg / m2 performed by administering fludarabine at a dose of about 20 mg / m² per day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein lymphodepletion is performed for a total of five days.

[0377] In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of about 50 mg / m² per day for two days and fludarabine at a dose of about 20 mg / m² 2 per day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein lymphodepletion is performed for a total of five days. 2 performed by administering fludarabine at a dose of about 20 mg / m² per day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein lymphodepletion is performed for a total of five days.

[0378] In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of about 40 mg / m² 2 per day for two days and fludarabine at a dose of about 20 mg / m² 2 per day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein lymphodepletion is performed for a total of five days.

[0379] In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of about 40 mg / m² 2 per day for two days and fludarabine at a dose of about 15 mg / m² 2 per day for five days, wherein cyclophosphamide and fludarabine are both administered on the first two days, and wherein lymphodepletion is performed for a total of five days.

[0380] In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg / m² 2 per day and fludarabine at a dose of 25 mg / m² 2 per day for two days, followed by administering fludarabine at a dose of 25 mg / m² 2 per day for three days.

[0381] In some embodiments, cyclophosphamide is administered together with mesna. In some embodiments, mesna is administered at 15 mg / kg. In some embodiments of infusing mesna, and if continuous infusion, mesna can be infused with cyclophosphamide for about 2 hours (day -5 and / or day -4), and then the remaining 22 hours are infused at a rate of 3 mg / kg / hour within 24 hours, starting simultaneously with each cyclophosphamide dose.

[0382] In some embodiments, lymphodepletion includes the step of treating the patient with an IL-2 regimen starting on the second day after administering the third TIL population to the patient.

[0383] In some embodiments, lymphodepletion includes the step of treating the patient with an IL-2 regimen starting on the same day as administering the third TIL population to the patient.

[0384] In some embodiments, lymphodepletion includes a 5-day pretreatment regimen. In some embodiments, the number of days is represented as days -5 to -1, or days 0 to 4. In some embodiments, the regimen includes administering cyclophosphamide on days -5 and -4 (i.e., days 0 and 1). In some embodiments, the regimen includes intravenously administering cyclophosphamide on days -5 and -4 (i.e., days 0 and 1). In some embodiments, the regimen includes intravenously administering cyclophosphamide at 60 mg / kg on days -5 and -4 (i.e., days 0 and 1). In some embodiments, cyclophosphamide is administered together with mesna. In some embodiments, the regimen further includes fludarabine. In some embodiments, the regimen further includes intravenously administering fludarabine. In some embodiments, the regimen further includes intravenously administering fludarabine at 25 mg / m 2 Intravenously administering fludarabine. In some embodiments, the regimen further includes intravenously administering fludarabine at 25 mg / m 2 Intravenously administering fludarabine. In some embodiments, the regimen further includes intravenously administering fludarabine at 25 mg / m 2 Intravenously administering fludarabine.

[0385] In some embodiments, a non-myeloablative lymphodepletion regimen includes the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day and administering fludarabine at a dose of 25 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for five days.

[0386] In some embodiments, a non-myeloablative lymphodepletion regimen includes the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for five days.

[0387] In some embodiments, a non-myeloablative lymphodepletion regimen includes the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for three days.

[0388] In some embodiments, a non-myeloablative lymphodepletion regimen includes the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day and administering fludarabine at a dose of 25 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2Administer fludarabine at a dose of [dose] per day for three days.

[0389] In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day and administering fludarabine at a dose of 25 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for one day.

[0390] In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for three days.

[0391] In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of: administering cyclophosphamide at a dose of 60 mg / m 2 / day and administering fludarabine at a dose of 25 mg / m 2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for three days.

[0392] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 4.

[0393] Table 4. Exemplary lymphodepletion and treatment regimens.

[0394]

[0395]

[0396] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 5.

[0397] Table 5. Exemplary lymphodepletion and treatment regimens.

[0398] Number of days -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 60 mg / kg X X Mesna (if needed) X X <![CDATA[Fludarabine 25 mg / m 2 / day]]> X X X X TIL infusion X

[0399] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 6.

[0400] Table 6. Exemplary lymphodepletion and treatment regimens.

[0401] Number of days -3 -2 -1 0 1 2 3 4 Cyclophosphamide 60 mg / kg X X Mesna (if needed) X X <![CDATA[Fludarabine 25 mg / m 2 / day]]> X X X TIL infusion X

[0402] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 7.

[0403] Table 7. Exemplary lymphodepletion and treatment regimens.

[0404] Number of days -5 -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 60 mg / kg X X Mesna (if needed) X X <![CDATA[Fludarabine 25 mg / m 2 / day]]> X X X TIL infusion X

[0405] In some embodiments, a non-myeloablative lymphodepletion regimen is administered according to Table 8.

[0406] Table 8. Exemplary lymphodepletion and treatment regimens.

[0407]

[0408]

[0409] In some embodiments, a non-myeloablative lymphodepletion regimen is administered according to Table 9.

[0410] Table 9. Exemplary lymphodepletion and treatment regimens.

[0411] Number of days -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 300 mg / kg X X Mesna (if needed) X X <![CDATA[Fludarabine 30 mg / m 2 / day]]> X X X X TIL infusion X

[0412] In some embodiments, a non-myeloablative lymphodepletion regimen is administered according to Table 10.

[0413] Table 10. Exemplary lymphodepletion and treatment regimens.

[0414] Number of days -3 -2 -1 0 1 2 3 4 Cyclophosphamide 300 mg / kg X X Mesna (if needed) X X <![CDATA[Fludarabine 30mg / m 2 / day]]> X X X TIL infusion X

[0415] In some embodiments, a non-myeloablative lymphodepletion regimen is administered according to Table 11.

[0416] Table 11. Exemplary lymphodepletion and treatment regimens.

[0417] Number of days -5 -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 300 mg / kg X X Mesna (if needed) X X <![CDATA[Fludarabine 30 mg / m 2 / day]]> X X X TIL infusion X

[0418] In some embodiments, the TIL infusion used in conjunction with the foregoing embodiments of the myeloablative lymphodepletion regimen can be any TIL composition described herein, as well as the addition of an IL-2 regimen and the administration of combination therapies (such as PD-1 and PD-L1 inhibitors) as described herein.

[0419] 2. IL-2 regimen

[0420] In some embodiments, the IL-2 regimen includes a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen includes intravenous administration of aldesleukin or a biosimilar or variant thereof starting on the second day following administration of a therapeutically effective portion of the therapeutic TIL population, wherein aldesleukin or a biosimilar or variant thereof is administered at a dose of 0.037 mg / kg or 0.044 mg / kg IU / kg (patient body weight), by intravenous infusion as a 15-minute bolus every eight hours until tolerance, up to 14 doses. After a 9-day rest, this schedule may be repeated for an additional 14 doses, for a total of up to 28 doses. 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 dose of up to 6 doses.

[0421] In some embodiments, the IL-2 regimen includes a tapered IL-2 regimen. The tapered IL-2 regimen has been described in O’Day et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In some embodiments, the tapered IL-2 regimen includes intravenous administration of 18 × 10 6 IU / m 2 aldesleukin or a biosimilar or variant thereof over 6 hours, followed by intravenous administration of 18 × 10 6 IU / m 2 over 12 hours, followed by intravenous administration of 18 × 10 6 IU / m 2 over 24 hours, followed by intravenous administration of 4.5 × 10 6 IU / m 2 . This treatment cycle may be repeated every 28 days for up to four cycles. In some embodiments, the tapered IL-2 regimen includes 18,000,000 IU / m 2 on day 1, 9,000,000 IU / m 2 on day 2, and 4,500,000 IU / m 2 on days 3 and 4.

[0422] In some embodiments, the IL-2 regimen includes a low-dose IL-2 regimen. Any low-dose IL-2 regimen known in the art can be used, including those described in Dominguez-Villar and Hafler, Nat. Immunol. 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, the low-dose IL-2 regimen includes administering 18×10 6 IU / m 2 aldesleukin or a biosimilar or variant thereof by continuous infusion every 24 hours for 5 days, followed by 2 days to 6 days without IL-2 therapy, optionally followed by administering 18×10 6 IU / m 2 intravenous aldesleukin or a biosimilar or variant thereof by continuous infusion for an additional 5 days, optionally followed by 3 weeks without IL-2 therapy, after which additional cycles can be administered.

[0423] In some embodiments, IL-2 is administered at a maximum dose of up to 6 doses. In some embodiments, the high-dose IL-2 regimen is suitable for pediatric use. In some embodiments, aldesleukin is used at a dose of 600,000 international units (IU) / kg every 8 hours to 12 hours, up to 6 doses. In some embodiments, aldesleukin is used at a dose of 500,000 international units (IU) / kg every 8 hours to 12 hours, up to 6 doses. In some embodiments, aldesleukin is used at a dose of 400,000 international units (IU) / kg every 8 hours to 12 hours, up to 6 doses. In some embodiments, aldesleukin is used at a dose of 500,000 international units (IU) / kg every 8 hours to 12 hours, up to 6 doses. In some embodiments, aldesleukin is used at a dose of 300,000 international units (IU) / kg every 8 hours to 12 hours, up to 6 doses. In some embodiments, aldesleukin is used at a dose of 200,000 international units (IU) / kg every 8 hours to 12 hours, up to 6 doses. In some embodiments, aldesleukin is used at a dose of 100,000 international units (IU) / kg every 8 hours to 12 hours, up to 6 doses.

[0424] In some embodiments, the IL-2 regimen comprises administering pegylated IL-2 at a dose of from 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bempegaldesleukin or a fragment, variant, or biosimilar thereof at a dose of from 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days.

[0425] In some embodiments, the IL-2 regimen comprises administering THOR-707 or a fragment, variant, or biosimilar thereof at a dose of from 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days.

[0426] In some embodiments, the IL-2 regimen comprises administering nelotanserin alpha or a fragment, variant, or biosimilar thereof after administration of TIL. In certain embodiments, nelotanserin is administered to a patient at a dose of from 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days.

[0427] In some embodiments, the antibody-cytokine transbody described herein has a longer serum half-life than wild-type IL-2 molecules (such as but not limited to aldesleukin or comparable molecules).

[0428] In some embodiments, the TIL infusion used in conjunction with the foregoing embodiments of the myeloablative lymphodepletion regimen can be any of the TIL compositions described herein, and can also include an infusion of MIL and PBL in place of the TIL infusion, as well as the addition of an IL-2 regimen and the administration of combination therapies as described herein (such as PD-1 and / or PD-L1 inhibitors and / or CTLA-4 inhibitors). Examples

[0429] The embodiments contained herein are now described with reference to the following examples. These examples are provided for illustrative purposes only, and the disclosure covered herein should in no way be construed as being limited by these examples, but rather should be construed as covering any and all variations that become apparent as a result of the teachings provided herein.

[0430] Example 1: Exemplary method for manufacturing TIL for gene editing

[0431] Figure 1A- An exemplary TIL manufacturing process is depicted in Figure 1F. Briefly, on Day 0, tumor tissue is isolated in a cryogenic solution, the bioburden sample is stored in a transport medium, and tumor fragments are inoculated into multiple (2, 3, or 4) G-Rex 100MCS flasks at a density of ≤50 fragments / flask. The excess fragments are snap-frozen. In some embodiments, the activation step can be incorporated into the preREP step, which provides a better co-stimulatory environment for TILs within rosettes / tumor MEs. For example, on Day 3, 60 μg of OKT3 or TransAct is added to each of the multiple G-Rex 100MCS flasks, and the cells are activated for the first time. On Day 7 / 8, the volume is reduced, the sample is filtered and transferred to a pooled EXP1000. The sample is removed for cell counting / viability analysis. The cells are washed, centrifuged at 400 g at 20 °C for 10 min, and divided into TALEN samples and control samples at a ratio of ≥9:1. The cells are resuspended in T buffer and electroporated with TALEN mRNA (TALEN samples) or no RNA (control samples) in a 10x10 6 / cuvette. The electroporated control and TALEN samples from each cuvette are inoculated into G-Rex100M flasks and incubated at 37 °C for 1 hour, which are filled with 100 mL of medium containing 6000 IU / mL IL-2. The feeder cells are irradiated, thawed, pooled, and incubated with IL-2. The sample is removed for cell counting and viability analysis. The feeder cells and additional IL-2 and OKT3 are added to the incubated control and TALEN samples in the G-Rex 100MCS flasks to generate REP cultures (1x G-Rex 100MCS for the control REP culture and ≤9x G-Rex 100MCS for the TALEN REP culture). On Day 10 / 11, 500 mL of medium and 3000 IU / mL IL-2 are added to the flasks of each REP culture. Alternatively, the cell medium can be completely replaced with fresh medium. This step does not require transferring the cell suspension and does not use G-Rex 500MCS. On Day 16, the volume is reduced, and the samples are pooled. The samples are transferred through a blood filter, and the samples are removed for cell counting / viability analysis. The control samples are centrifuged, and a control final formulation is generated and cryopreserved under controlled-rate freezing. The TALEN samples are processed through a LOVO system, and a TALEN retentate final formulation is generated and cryopreserved under controlled-rate freezing. Then, quality control analysis is performed on the formulated product samples.

[0432] This process has the following advantages: a) The step of transferring the suspension for activation in the bag is removed; b) Allows a 1 / 10 scale control; c) The transfer during the process to G-Rex10 is removed; d) The overnight incubation step at 30°C is removed, facilitating immediate reactivation (REP) with ambient temperature medium; e) One treatment day is removed.

[0433] Example 2: Shortened proliferation assay of TILs for gene editing

[0434] Currently, the IL-2 independent proliferation assay of TILs for gene editing takes 28 days. A study was conducted to determine whether the current IL-2 independent proliferation assay could shorten the time period to approximately 14 days or less.

[0435] Experimental design

[0436] Two TALEN-edited TIL products at 18 and 22 days were amplified from tumor samples L4346 and M1214 and seeded at 2e6 cells / well in a G-Rex 24-well plate on day 0 and cultured in cell medium composed of RPMI+Glutamax (50%), AIM V (50%), human AB serum (10%), gentamicin (0.1%), and 2-mercaptoethanol (0.1%) with or without IL-2 (300 IU / mL). The cell numbers were counted on days 7, 10, and 14 after the start of cell culture.

[0437] Results

[0438] Figure 2 The proliferation of TILs (expressed as fold expansion) is shown: TILs cultured in the presence of IL-2 continued to proliferate after inoculation, while TILs cultured in the absence of IL-2 had little increase in fold expansion on day 7, but in both experiments, the cell numbers decreased by day 14 (<1 fold expansion).

[0439] Example 3: Click-iT using Jurkat cells TM EdU proliferation assay

[0440] A preliminary study was conducted to test the Click-iT TM EdU proliferation assay conditions for Jurkat cells.

[0441] Experimental design

[0442] On day 0, Jurkat cells were seeded at 1e4 cells / mL, 1e5 cells / mL, 2e5 cells / mL, and 1e6 cells / mL and the cells were cultured in the absence of IL-2. At 1 day, 5 days, and 7 days after the start of the culture, various concentrations of EdU (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0 μM) were added at multiple time points (24 hours, 8 hours, 6 hours, 4 hours, and 2 hours) prior to the Click-iT TM EdU proliferation assay.

[0443] After incubation with EdU, 80 μl of the culture medium was removed from each well and 50 μl of EdU fixative was added to each well. The plate was incubated at room temperature for 5 minutes. The fixative was removed and the wells were washed with 200 μl of EdU wash buffer. Then 50 μl of EdU reaction mixture (containing deionized water, EdU master mix, EdU reaction additive, and HRP-azide) was added to each well and the plate was incubated at room temperature for 30 minutes. The reaction mixture was removed and the plate was patted dry on a paper towel. Next, 200 μl of 1.5% BSA blocking solution was added to each well and the plate was incubated in the dark at room temperature for 5 minutes. The blocking solution was removed and the plate was washed 3 times with 200 μl of EdU wash buffer. 100 μl of Amplex TM UltraRed reaction buffer, Amplex TM UltraRed reagent, and hydrogen peroxide solution was added to each well and the plate was incubated in the dark at room temperature for 15 minutes. The reaction was terminated by adding 10 μl / well of Amplex TM UltraRed stop solution. Then the plate was read on a fluorescence microplate reader. TM

[0444] Results

[0445] Figure 3 and Figure 4 showed that at all cell concentrations, proliferation signals were observed in the presence of 10 - 20 μM EdU, where 1e3 cells / well - 1e4 cells / well was the optimal seeding condition.

[0446] Example 4: Click-iT TM EdU proliferation assay for gene-edited TILs

[0447] A study was conducted to test the Click-iT TM EdU proliferation assay conditions for gene-edited TILs.

[0448] Experimental design

[0449] Two PD-1 TALEN-edited TIL lines (L4340 and EP11231) were tested using the Click-iT TM EdU proliferation assay. The corresponding mock gene-edited TIL lines were used as negative controls, while Jurkat cells were used as positive controls.

[0450] On day 0, TILs were seeded in triplicate at 1e5 / well and 2e5 / well, and Jurkat cells were seeded in triplicate at 2e3 / well in cell culture medium without IL-2. Serial dilutions of Jurkat cells at 4e5 / well, 1e5 / well, 2.5e4 / well, 6.25e4 / well, and 1.56e4 / well were also tested.

[0451] The Click-iT TM EdU proliferation assay was performed on days 1, 7, 10, and 14 after seeding. EdU at concentrations of 0 μM, 10 μM, 20 μM, and 40 μM was added 24 hours before running the Click-iT TM EdU proliferation assay.

[0452] Results

[0453] Figure 5 showed that for the two PD-1 TALEN-edited TIL lines and the mock gene-edited TIL lines, no proliferation was observed on days 7, 10, and 14.

[0454] Example 5: Click-iT TM EdU proliferation assay for additional gene-edited TIL batches

[0455] A further study was conducted to test the Click-iT TM EdU proliferation assay conditions for an additional five gene-edited TIL batches.

[0456] Experimental design

[0457] Five PD-1 TALEN-edited TIL batches (L4374, K7091, L4353, L4340, and EP11231) were tested using the Click-iT TM EdU proliferation assay. Jurkat cells were used as positive controls.

[0458] On day 0, TILs were seeded in triplicate at 1e5 cells / well and Jurkat cells were seeded in triplicate at 2e3 cells / well in cell culture medium without IL-2.

[0459] Click-iT was performed on days 7 and 10 after seeding. TM EdU proliferation assay. EdU was added at a concentration of 20 μM 24 hours before running the Click-iT TM EdU proliferation assay.

[0460] Results

[0461] Figure 6 showed that for five PD-1 TALEN-edited TIL batches, no proliferation was observed on days 7 and 10.

[0462] The above-described examples are provided to give those of ordinary skill in the art a complete disclosure and description of embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope that the inventors regard as their invention. Modifications to the above-described modes for carrying out the invention that are obvious to those of skill in the art are intended to fall within the scope of the appended claims. All patents and publications mentioned in this specification indicate the level of skill of those in the art to which the present invention pertains.

[0463] All headings and section names are used for clarity and reference purposes only and should not be regarded as limiting in any way. For example, those skilled in the art will appreciate that it may be useful to combine aspects from different headings and sections depending on the spirit and scope of the present invention as described herein.

[0464] All references cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0465] Many modifications and variations can be made to this application without departing from the spirit and scope of the application, which will be obvious to those of skill in the art. The specific embodiments and examples described herein are provided by way of illustration only, and this application is limited only by the terms of the appended claims and the full scope of equivalents of those claims.

Claims

1. A method for evaluating the proliferation index of a population of gene - edited T cells, the method comprising: (a) Culturing a first portion of the population of gene - edited T cells in a cell culture medium without IL - 2; (b) Adding an agent to the cell culture medium, wherein the agent is incorporated into the DNA of the cells during proliferation; (c) Measuring the amount of the agent incorporated into the gene - edited T cells at one or more time points; and (d) Calculating the proliferation index of the population of gene - edited T cells based on the amount of the agent incorporated into the gene - edited T cells at the one or more time points.

2. The method according to claim 1, wherein the agent is a modified nucleotide selected from the group consisting of: EdU (5-ethynyl-2'-deoxyuridine), 5-ethynyluridine (5-EU), F-ara-EdU, bromo-2'-deoxyuridine (BrdU), and 3 H] thymidine ( 3 H] TdR).

3. The method according to claim 1 or 2, wherein the agent is EdU.

4. The method according to any one of claims 1 to 3, wherein measuring the amount of the agent incorporated into the T cells comprises performing a click reaction using HRP.

5. The method according to claim 4, the method further comprising adding Amplex UltraRed reagent, wherein the Amplex UltraRed is converted by the HRP into a fluorescent product.

6. The method according to claim 5, the method further comprising measuring the amount of the fluorescent product using a fluorescence reader.

7. The method according to any one of claims 1 to 6, wherein the one or more time points are selected from the group consisting of: day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, and day 28.

8. The method according to any one of claims 1 to 6, wherein the one or more time points include day 1, day 7, day 10, and day 14.

9. The method according to any one of claims 4 to 6, wherein the agent is added on day 0, day 6, day 9, and / or day 13.

10. The method according to any one of claims 1 to 9, wherein the agent is added 24 hours before measuring the amount of the agent incorporated into the gene - edited T cells.

11. The method according to any one of claims 1 to 10, wherein the agent is added at 0 uM, 10 uM, 20 uM, and / or 40 uM.

12. The method according to any one of claims 1 to 11, the method comprising culturing a second portion of the population of gene - edited T cells in the cell culture medium with IL - 2 as a positive control.

13. The method according to any one of claims 1 to 11, the method comprising culturing a population of transformed T cells in the cell culture medium without IL - 2 as a positive control.

14. The method according to claim 13, wherein the transformed T cells are Jurkat cells.

15. The method according to any one of claims 1 to 14, wherein the gene-edited T cells are gene-edited tumor-infiltrating lymphocytes (TILs) or CART cells.

16. The method according to claim 15, wherein the gene-edited T cells are gene-edited TILs.

17. The method according to claim 15 or 16, wherein the gene-edited TILs comprise a TALE nuclease system for regulating the expression of at least one protein.

18. The method according to claim 17, wherein the TALE nuclease system regulates the expression of PD-1.

19. The method according to claim 17, wherein the TALE nuclease system regulates the expression of CTLA-4.

20. The method according to claim 17, wherein the TALE nuclease system regulates the expression of LAG-3.

21. The method according to claim 17, wherein the TALE nuclease system regulates the expression of CISH.

22. The method according to claim 17, wherein the TALE nuclease system regulates the expression of CBL-B.

23. The method according to claim 17, wherein the TALE nuclease system regulates the expression of TIGIT.

24. The method according to claim 15 or 16, wherein the gene-edited TILs comprise a first TALE nuclease system for regulating the expression of a first protein and a second TALE nuclease system for regulating the expression of a second protein.

25. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and / or CBL-B.

26. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CTLA-4.

27. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and LAG-3.

28. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CISH.

29. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and CBL-B.

30. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of PD-1 and TIGIT.

31. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and LAG-3.

32. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CISH.

33. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CTLA-4 and CBL-B.

34. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CISH.

35. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of LAG-3 and CBL-B.

36. The method according to claim 24, wherein the first TALE nuclease system and the second TALE nuclease system regulate the expression of CISH and CBL-B.

37. The method according to claim 15, wherein the gene-edited T cells are CAR T cells.

38. The method according to claim 37, wherein the CAR of the CART cells is specific for an antigen selected from the group consisting of: CD19; CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule 1, CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG 72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin 13 receptor subunit alpha-2; mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen 4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme; Prostatic acid phosphatase (PAP); Mutated elongation factor 2 (ELF2M); Ephrin B2; Fibroblast activation protein α (FAP); Insulin-like growth factor 1 receptor (IGF-I receptor), Carbonic anhydrase IX (CAIX); Proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); Glycoprotein 100 (gp100); Oncogenic fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog (Abl) (bcr-abl); Tyrosinase; Ephrin type-A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3; Transglutaminase 5 (TGS5); High molecular weight melanoma-associated antigen (HMWMAA); O-acetyl GD2 ganglioside (OAcGD2); Folate receptor beta; Tumor endothelial marker 1 (TEM1 / CD248); Tumor endothelial marker 7-related (TEM7R); Claudin 6 (CLDN6); Thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor family C group 5 member D (GPRC5D); Chromosome X open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide moiety of globoH glycosphingolipid (GloboH); Breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); Adrenergic receptor beta-3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 on chromosome 12p (ETV6-AML); Spermatid protein 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein p53 (p53); p53 mutant; Prostein; Survivin; Telomerase; Prostate cancer tumor antigen 1; Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene);N-acetylglucosaminyltransferase V (NA17); Paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1 B1); CCCTC-binding factor (zinc finger protein)-like, squamous cell carcinoma antigen 3 recognized by T cells (SART3); Paired box protein Pax-5 (PAX5); Pre-acrosomal protein-binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase (LCK); Kinase anchor protein 4 (AKAP-4); Synovial sarcoma, X breakpoint 2 (SSX2); Receptor for advanced glycation end products (RAGE-1); Renal ubiquitin 1 (RU1); Renal ubiquitin 2 (RU2); Legumain; Human papillomavirus E6 (HPV E6); Human papillomavirus E7 (HPV E7); Intestinal carboxylesterase; Mutant heat shock protein 70-2 (muthsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Phosphatidylinositol proteoglycan 3 (GPC3); Fc receptor-like 5 (FCRL5); And immunoglobulin lambda-like polypeptide 1 (IGLL1).

39. A method for treating a cancer patient, the method comprising: (a) generating a gene-edited TIL population from a tumor excised from the cancer patient; (b) evaluating the proliferation index of the gene-edited TIL population using the method according to any one of claims 1 to 36; and (c) If the proliferation index of the gene-edited TIL population is lower than that of the positive control, administer a therapeutically effective dose of the gene-edited TILs to the cancer patient.

40. The method according to claim 39, wherein the cancer is selected from the group consisting of: melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

41. The method according to claim 39 or 40, wherein the therapeutically effective dose of the gene-edited TILs is from about 1×10 9 to about 1×10 11 TILs.

42. The method according to any one of claims 39 to 41, wherein a non-myeloablative lymphodepletion regimen has been administered to the patient prior to administering the therapeutically effective dose of the gene-edited TILs to the patient in step (c).

43. The method according to any one of claims 39 to 42, the method further comprising the step of treating the patient with a high-dose IL-2 regimen starting on the day after administering the therapeutically effective dose of the gene-edited TILs to the patient in step (c).

44. The method according to any one of claims 39 to 43, wherein the cancer is melanoma.

45. The method according to claim 44, wherein the cancer is metastatic melanoma.

46. The method according to any one of claims 39 to 43, wherein the cancer is NSCLC.

47. The method according to claim 46, wherein the cancer is metastatic NSCLC.

48. The method according to any one of claims 39 to 47, wherein the gene editing causes the expression of one or more immune checkpoint genes in at least a portion of the gene-edited TIL population to be silenced or reduced.

49. The method according to any one of claims 39 to 48, the method further comprising the method according to any one of claims 1 to 38.

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