Methods for assessing the proliferative potential of gene-edited T cells

Abstract

The present invention provides improved methods for assessing the proliferative potential of gene-edited T cells, such as, for example, gene-edited tumor-infiltrating lymphocytes (TILs), CART cells, etc.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent No. 63 / 384,598, filed November 21, 2022, all of which are incorporated herein by reference in their entirety. [Background technology]

[0002] TALEN gene editing has demonstrated highly specific and durable gene disruption in both human and mouse T cells. However, if TALENs remain active for extended periods, off-site target cleavage may occur. These off-site modifications in cells may contribute to cell transformation. Interleukin-2 (IL-2) is a cytokine required for T cell proliferation. IL-2-independent growth indicates cell transformation and can be considered cancerous.

[0003] The present invention provides a shortened process and method for assessing the proliferative potential of gene-edited T cells, such as TILs and CART cells. Summary of the Invention

[0004] Provided herein are improved methods for assessing the proliferative potential of gene-edited tumor-infiltrating lymphocytes (TILs), gene-edited T cells, such as CART cells.

[0005] In some embodiments, provided herein are methods for assessing the proliferation index of a gene-edited T cell population, the method comprising: (a) culturing a first portion of the gene-edited T cell population in cell culture medium that does not contain IL-2; (b) adding an agent to the cell culture medium, where the agent is incorporated into the DNA of the cells during proliferation; (c) measuring the amount of agent incorporated into the gene-edited T cells at one or more time points; and (d) calculating the proliferation index of the gene-edited T cell population by the amount of agent incorporated into the gene-edited T cells at the one or more time points.

[0006] In some embodiments, the agent is 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 ([ 3In some embodiments, the drug is EdU. In some embodiments, measuring the amount of drug incorporated into the T cells comprises performing a click reaction using HRP. In some embodiments, the method further comprises adding Amplex UltraRed reagent, wherein Amplex UltraRed is converted to a fluorescent product by HRP. In some embodiments, the method further comprises measuring the amount of fluorescent product using a fluorescence reader. In some embodiments, the one or more time points are selected from the group consisting of: 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 day, and 28 day. In some embodiments, the one or more time points comprise 1 day, 7 day, 10 day, and 14 day. 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 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 comprises culturing a second portion of the gene-edited T cell population in cell culture medium containing IL-2 as a positive control. In some embodiments, the method further comprises culturing a 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.

[0007] In some embodiments, the gene-edited T cells are gene-edited tumor-infiltrating lymphocytes (TILs) or CART 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 expression of at least one protein. In some embodiments, the TALE-nuclease system regulates expression of PD-1. In some embodiments, the TALE-nuclease system regulates expression of CTLA-4. In some embodiments, the TALE-nuclease system regulates expression of LAG-3. In some embodiments, the TALE-nuclease system regulates expression of CISH. In some embodiments, the TALE-nuclease system regulates expression of CBL-B. In some embodiments, the TALE-nuclease system regulates expression of TIGIT. In some embodiments, the gene-edited TILs comprise a first TALE-nuclease system for regulating expression of a first protein and a second TALE-nuclease system for regulating 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 CART cells. In some embodiments, the CAR of the CART cell is CD19; CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule-1,CD33; epidermal growth factor receptor variant II (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tn antigen ((TnAg) 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); CD 20; 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); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropein) subunit, beta,9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (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 class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary 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 K9 (LY6K); olfactory receptor 51 E2 (OR51 E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAG E-1 a); melanoma-associated antigen 1 (MAG E-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm 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; surviving; telomerase; prostate cancer tumor antigen-1,Melanoma antigen 1 recognized by T cells; rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA1 7); 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 1 B1 (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); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (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).

[0008] Further provided herein is a method for treating a cancer patient, the method comprising: (a) generating a gene-edited TIL population from a tumor resected from the cancer patient; (b) assessing the proliferation index of the gene-edited TIL population using the method of any one of claims 1-36; and (c) administering a therapeutically effective dose of 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 positive control.

[0009] 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, cancer 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 gene-edited TILs is about 1 x 10 9 ~Approx. 1×10 11 In some embodiments, the patient is a TIL. In some embodiments, prior to administering a therapeutically effective dose of the gene-edited TIL cells to the patient in step (c), the patient has been administered a non-myeloablative lymphodepletion regimen. In some embodiments, the method further comprises treating the patient with a high-dose IL-2 regimen starting the day after administering a therapeutically effective dose of the gene-edited TIL cells 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 silences or reduces expression of one or more immune checkpoint genes in at least a portion of the gene-edited TIL population. [Brief explanation of the drawings]

[0010] [Figure 1A] An exemplary manufacturing process for gene-edited TILs. [Figure 1B] An exemplary manufacturing process for gene-edited TILs. [Figure 2] Fold expansion time course of TILs in the presence and absence of IL-2. [Figure 3] Proliferation profiles of Jurkat cells at various seeding densities, multiple time points (days 1, 5, and 7), and multiple EdU concentrations. [Figure 4] Proliferation profiles of Jurkat cells at various seeding densities and multiple intervals for EdU addition. [Figure 5] Growth profiles of two gene-edited TIL lots at multiple time points (days 1, 7, 10, and 14). [Figure 6] Growth profiles of five gene-edited TIL lots at multiple time points (days 7 and 10).

[0011] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0012] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0013] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0014] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0015] SEQ ID NO: 5 is an IL-2 form.

[0016] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0017] SEQ ID NO: 7 is an IL-2 form.

[0018] SEQ ID NO: 8 is a mucin domain polypeptide.

[0019] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0020] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0021] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0022] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0023] SEQ ID NO: 13 is the target PD-1 sequence.

[0024] SEQ ID NO: 14 is the target PD-1 sequence.

[0025] SEQ ID NO: 15 is the repeated PD-1 left repeat sequence.

[0026] SEQ ID NO: 16 is a repeated PD-1 right repeat sequence.

[0027] SEQ ID NO: 17 is the repeated PD-1 left repeat sequence.

[0028] SEQ ID NO: 18 is a repeated PD-1 right repeat sequence.

[0029] SEQ ID NO: 19 is the PD-1 left TALEN nuclease sequence.

[0030] SEQ ID NO: 20 is the PD-1 right TALEN nuclease sequence.

[0031] SEQ ID NO: 21 is the PD-1 left TALEN nuclease sequence.

[0032] SEQ ID NO: 22 is the PD-1 right TALEN nuclease sequence.

[0033] SEQ ID NO: 23 is the amino acid sequence of an exemplary Clo05 1 nuclease domain.

[0034] I. Definition 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 referenced herein are incorporated by reference in their entirety.

[0035] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Simultaneous administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0036] The term "in vivo" refers to events that take place inside a subject's body.

[0037] The term "in vitro" refers to events that occur outside a subject's body. In vitro assays include cell-based assays, in which living or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0038] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic manner.

[0039] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) over a period of one week, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a period of one week, or most preferably at least about 100-fold over a period of one week. Several rapid expansion protocols are described herein.

[0040] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells initially obtained as leukocytes that have left the bloodstream of a subject and migrated into a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0041] As used herein, a "cell population" (including TILs) refers to a large number of cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically yields a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0042] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0043] By "thawed cryopreserved TILs" herein is meant a population of TILs that have previously been cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or the temperature at which the TILs can be administered to a patient.

[0044] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence therapy. TILs can generally be classified by 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 upon reintroduction into patients.

[0045] 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% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium can be referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium that contains DMSO. In some embodiments, CS10 medium contains 10% DMSO.

[0046] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0047] 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.

[0048] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0049] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0050] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA), and muromonab, or a variant, conservative amino acid substitution, glycoform, or biosimilar thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0051] [Table 1]

[0052] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. 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 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4).

[0053] [Table 2-1] [Table 2-2]

[0054] 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 as well as 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. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several 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 shown in Table 2 (SEQ ID NO: 9).

[0055] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several 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 a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0056] 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, including human and mammalian forms, conservative amino acid substitutions, glycoforms, 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 β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several 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 shown in Table 2 (SEQ ID NO: 11).

[0057] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, 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 primarily stimulates natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several 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 shown in Table 2 (SEQ ID NO: 12).

[0058] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, the tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg of body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10The TIL (optionally including genetically engineered TIL) compositions may be administered at doses of 1000-150 ...

[0059] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0060] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0061] The term "microenvironment" as used herein may refer to the microenvironment of a solid or hematological tumor as a whole, or to individual subsets 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 promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0062] In some embodiments, the invention includes methods of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27-23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg intravenously every 8 hours to physiological tolerance.

[0063] Experimental results indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy 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 referred to as "immunosuppressive conditioning") in patients prior to introducing the TILs of the present invention.

[0064] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to accomplish its intended purpose, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), or the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the mode of administration. The term also applies to a dose that induces a specific response in target cells (e.g., reduced 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 timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0065] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment," as used herein, encompasses any treatment of disease in mammals, particularly humans, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also meant to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0066] The terms "non-myeloablative chemotherapy," "non-myeloablative lymphodepletion," "NMALD," "NMA LD," "NMA-LD," and any variants of the foregoing, are used interchangeably to refer to chemotherapy regimens designed to deplete a patient's lymphoid immune cells while avoiding depletion of the patient's myeloid immune cells. Typically, a patient undergoes a course of non-myeloablative chemotherapy before administering tumor-infiltrating lymphocytes to the patient, as described herein.

[0067] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0068] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs 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. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and 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. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0069] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein containing 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 specific 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 include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0070] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells initially obtained as leukocytes that have left the bloodstream of a subject and migrated into a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, a second expanded TIL or a second additional expanded TIL.

[0071] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence therapy. TILs can generally be classified by 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 upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their 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. For example, TILs can be considered potent if 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, or greater than about 1000 pg / mL.

[0072] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, the base moiety, and / or the linkages between deoxyribonucleotides in an oligonucleotide.

[0073] 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 a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, 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 can 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.

[0074] 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 inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0075] The terms "about" and "approximately" refer to values ​​within a statistically meaningful range. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, and even more preferably within 5%. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may be used with the described configurations.

[0076] The transitional phrases "comprising," "consisting essentially of," and "consisting of," when used in the appended claims, in their original and amended forms, define the claims in terms of what additional unrecited claim elements or steps, if any, are excluded from the claim(s). 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, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the scope of the claim to the specified element, step, or material(s) and those that do not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0077] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain comprises a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions, termed framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0078] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound to a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen preferably reacts with a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0079] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0080] As used herein, the term "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 to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL and (vi) domain antibody (dAb) fragments, which may consist of two domains, V and VD (Ward, et al., Nature, 1989, 341, 544-546), and (vi) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, they can be synthesized using recombinant methods L and V H The domains may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L If the domain is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV LMethods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0081] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0082] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0083] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes 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, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0084] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0085] The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0086] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0087] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. 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).

[0088] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0089] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described in, for example, 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.

[0090] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, 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 those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation may increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these 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, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes may be used to cleave fucose residues from antibodies. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0091] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0092] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product that is similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, 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), a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product that is similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorization or licensing purposes under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Additionally, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo nonclinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may include an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in changes in the safety and / or efficacy of the pharmaceutical.A biosimilar may have the same or a different glycosylation pattern as the reference drug. In particular, but not exclusively, a biosimilar may have a different glycosylation pattern if the difference addresses or is intended to address safety concerns associated with the reference drug. Additionally, a biosimilar may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the safety and efficacy of the drug are not compromised. A biosimilar may contain differences, for example, in its pharmacokinetic (PK) and / or pharmacodynamic (PD) profile compared to the reference drug, but still be considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, a biosimilar may exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered a barrier to approval as a similar biological product by regulatory authorities, such as the EMA. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0093] The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, which, when present in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and 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") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with one another. In some embodiments, the set of polypeptides comprises a dimerization switch that, in the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., couple the antigen-binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta 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 costimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory 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 comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0094] II. Methods for assessing the proliferation index

[0003] Embodiments of the present invention relate to methods for assessing the proliferation index of a T cell population. In some embodiments, the T cells are gene-edited T cells. As used herein, "gene-editing," "gene editing," and "genome editing" refer to types of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified, or replaced within the genome of a cell. In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockdown) expression of a DNA sequence. In some embodiments, gene editing involves the expression of an exogenous protein in the cell, such as a chimeric antigen receptor (CAR).

[0095] In some embodiments, the method comprises: (a) culturing a first portion of the gene-edited T cell population in cell culture medium that does not contain IL-2; (b) adding an agent to the cell culture medium, where the agent is incorporated into the DNA of the cells during proliferation; (c) measuring the amount of drug incorporated into the gene-edited T cells at one or more time points; and (d) calculating a proliferation index of the gene-edited T cell population according to the amount of agent incorporated into the gene-edited T cells at one or more time points.

[0096] In some embodiments, the gene-edited T cells can be expanded in cell culture medium without 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 assessed after the expansion period. Any of several well-known methods can be used to assess the proliferation level of the gene-edited T cells (and corresponding controls). In various examples, the method may use a fluorescent reader. The use of a fluorescent reader readily allows for the detection of fluorescent signals that can be useful in assessing the proliferation level of the gene-edited T cells. For example, as further described below, characteristics directly related to indicative of the proliferation level can be detected.

[0097] The proliferation level of gene-edited T cells according to the methods of the present invention can be determined by any of several known proliferation assays. These assays can fall into one of the following categories, including assays involving (i) measurement of DNA synthesis, (ii) detection of proliferation-specific cell markers, (iii) measurement of successive cell divisions using cell membrane-binding dyes, (iv) measurement of cellular DNA content, and (v) measurement of cellular 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.

[0098] As described above, a DNA synthesis assay can be used to determine the proliferation level of gene-edited T cells using the methods of the present invention. In one example of such a method, the incorporation of non-radioactive modified nucleotides into the DNA of dividing cells is detected as a measure of proliferation. As an example, the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) can be used to assess active DNA synthesis. Such an analog (e.g., EdU) can be added to proliferating cells before the end of the proliferation period described above, for example, about 24 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, or about 2 hours before the end of the proliferation period. EdU can be added to cell culture medium to 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 cell culture medium to a concentration of about 20 μM.

[0099] The incorporated analog (e.g., EdU) can be detected, for example, by a click reaction (copper-catalyzed azide-alkyne cycloaddition) using a fluorescent probe (e.g., Amplex UltraRed reagent, Click-iT® EdU Alexa Fluor® 488, etc.), facilitating 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):241 5-20, 2008, the contents of which are incorporated herein by reference in their entireties).

[0100] In some embodiments, cells are seeded into 96-well plates on day 0. The number of TILs added per well can be 1e4, 1e5, 2e5, or 1e6. In some embodiments, 1e5 TILs are added per well. The number of Jurkat cells added per well can be 1e3, 2e3, or 1e4. In some embodiments, 1e3 Jurkat cells are added per well. In some embodiments, 2e3 Jurkat cells are added per well. In some embodiments, each sample can be run in duplicate, triplicate, quadruplicate, etc.

[0101] EdU (20 μM) is added to the appropriate wells 24 hours before performing the assay, for example, on day 6 and / or day 9. Measurements can be performed on day 7 and / or day 10. To perform the assay, 80 μl of medium is removed from each well, and 50 μl of Click-iT® 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 Click-iT® EdU wash buffer. 50 μl of Click-iT® EdU reaction cocktail, containing deionized water, Click-iT® EdU master mix, Click-iT® EdU reaction additive, and HRP-azide, is then added to each well and incubated at room temperature for 30 minutes. The reaction cocktail is removed from the plate. Then, 200 μl of 1.5% BSA blocking solution is added to each well and incubated at room temperature for 5 minutes, protected from light. The blocking solution is removed from the plate. The plate is then washed three times with 200 μl of Click-iT® EdU wash buffer. The reaction is initiated by adding 100 μl of Amplex™ UltraRed reaction mixture containing Amplex™ UltraRed reaction buffer, Amplex™ UltraRed reagent, and hydrogen peroxide solution, and the plate is incubated at room temperature protected from light for 15 minutes. The reaction is stopped by adding 10 μl / well of Amplex™ UltraRed stop solution. The plate is then read in a fluorescence microplate reader (excitation 568 nm and emission 585 nm are recommended).

[0102] In another example, 5-bromo-2'-deoxyuridine (BrdU) can be used to detect cell proliferation. When gene-edited T cells are cultured in a labeling medium containing BrdU, this pyrimidine analog is incorporated in place of thymidine in newly synthesized DNA. Detection of incorporated BrdU can be achieved using an anti-BrdU antibody (see, for example, Porstmann et al., J Immunol Methods 82(1):169-79, 1985).

[0103] Another example for detecting DNA synthesis is the use of radioactive nucleotides, [ 3 H]thymidine ([ 3 H]TdR) to attach to new strands of chromosomal DNA during cell division. 3 This involves detecting the incorporation of [H]TdR (see, e.g., Denton, Methods Mol Biol 79:1 69-77, 1998). 3 Radioactivity can be measured in DNA recovered from [H]TdR-treated cells.

[0104] As mentioned above, assays that detect proliferation-specific cell markers can also be utilized in the context of the present invention. One exemplary assay involves the detection of nuclear-specific proliferation antigen, Ki-67. The detection of Ki-67 protein expression in proliferating cells is achieved through the use of anti-Ki-67 antibodies, followed by either imaging techniques or flow cytometry (see, for example, Soares et al., J Immunol Methods 362(1-2):43-50, 2010). Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in quiescent cells.

[0105] In other examples, cell membrane-bound dyes can be utilized in the context of the present invention to assess cell proliferation. Some exemplary dyes that can be used include carboxyfluorescein succinimidyl ester (CFSE) and CellTrace™ Far Red. Such dyes cross the cell's plasma membrane, covalently bind to all free amines on the cell surface and interior, and can be retained for long periods of time. The fluorescent signal of the dye after incorporation into the cell can be detected by a fluorescence reader or flow cytometry. The probe signal can then be used to monitor proliferation by the progressive half-life of fluorescence in daughter cells after each cell division (see, e.g., Tario et al., J Vis Exp(70):e4287, 2012, and Filby et al., Methods 82:29-37, 2015).

[0106] Cellular DNA content can also be measured to determine the degree of cell proliferation. One exemplary method that can be used is the CyQUANT® cell proliferation assay, which uses a green fluorescent nucleic acid stain and a background suppression dye that is impermeable to live cells, inhibits the nucleic acid stain, and blocks the staining of dead cells and cells with compromised cell membranes. Detection of stained DNA is achieved by measuring fluorescence using a fluorescence reader (see, for example, Jones et al., J Immunol Methods 254(1-2):85-98, 2001).

[0107] In another example, cellular metabolism can be assessed in the context of the present invention to determine cell proliferation. Tetrazolium salts such as MTT and MTS can be used to assess cellular metabolic activity, which reflects the number of viable cells present in a sample. NAD(P)H-dependent cellular oxidoreductase enzymes can reduce such tetrazolium salts to colored, insoluble formazan dyes under defined conditions. The amount of colored product formed can be quantified by measuring light absorption at specific wavelengths through 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). A related compound for measuring cellular metabolic activity, alamarBlue®, has 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):21 1-24, 1994). In addition, ATP bioluminescence can be used as a measure of cell proliferation, correlating the concentration of ATP in a given sample with the number of viable cells (see, e.g., Crouch et al., J Immunol Methods 160(1):81-8, 1993).

[0108] The proliferation level of gene-edited T cells in the absence of IL-2 can be expressed as a number, such as a proliferation index (PI), which reflects the transformant state of the gene-edited T cells according to the methods of the present invention. This proliferation level of gene-edited T cells in the absence of IL-2 can be compared, for example, with the proliferation level of a positive control sample of a transformed cell line (e.g., Jurkat cells) that exhibits IL-2-independent growth, the proliferation level of a positive control sample of gene-edited T cells in the presence of IL-2, or both. Furthermore, the proliferation level of gene-edited T cells in the absence of IL-2 can be compared, for example, 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 these pieces of information, a proliferation index (PI) according to one of the following formulas can be used as an assessment measure of the transformation state of the gene-edited T cells: PI = (proliferation level of gene-edited T cells in the absence of IL-2), PI = (proliferation level of gene-edited T cells in the absence of IL-2) - (proliferation level of negative control sample in the absence of IL-2), PI = (proliferation level of gene-edited T cells in the absence of IL-2) / (proliferation level of positive control sample in the absence of IL-2), or PI = [(proliferation level of gene-edited T cells in the absence of IL-2) - (proliferation level of negative control sample in the absence of IL-2)] / (proliferation level of positive control sample in the absence of IL-2).

[0109] In some embodiments, the gene-edited T cells are gene-edited TILs.

[0110] In some embodiments, the gene-edited T cells are CARTs, which are T cells comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., an antibody or antibody fragment, a TCR or a TCR fragment) that binds to a tumor antigen described herein, a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) (e.g., an intracellular signaling domain comprising a costimulatory domain (e.g., a costimulatory domain described herein) and / or a primary signaling domain (e.g., a primary signaling domain described herein). CAR nucleic acid constructs, encoded proteins, containing vectors, host cells, pharmaceutical compositions, and administration and treatment methods related to the present invention are disclosed in detail in International Patent Application Publication No. WO2015 / 142675, which is incorporated by reference in its entirety.

[0111] III. Gene-edited TILs Embodiments of the present invention relate to gene-edited TIL populations, where the gene-edited TILs are produced using a method comprising one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, "gene editing," "gene editing," and "genome editing" refer to types of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified, or replaced within the genome of a cell. In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockdown) the expression of a DNA sequence. According to embodiments of the present invention, gene editing techniques are used to enhance the efficacy of therapeutic TIL populations.

[0112] The method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed 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 a further embodiment, the method for expanding TILs into a therapeutic TIL population is performed according to any embodiment of the methods described in WO2018 / 081473A1, WO2018 / 129332A1, or WO2018 / 182817A1, which are incorporated herein by reference in their entireties, wherein the method further comprises gene editing at least a portion of the TILs. Thus, certain 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 that is transferred to an infusion bag is permanently gene edited.

[0113] In some embodiments, the gene-edited TIL population comprises reduced expression of a protein.

[0114] In some embodiments, the protein is PD-1.

[0115] In some embodiments, the protein is CTLA-4.

[0116] In some embodiments, the protein is LAG-3.

[0117] In some embodiments, the protein is CISH.

[0118] In some embodiments, the protein is TIGIT.

[0119] In some embodiments, the protein is CBL-B.

[0120] In some embodiments, the gene-edited TIL population comprises reduced expression of a first protein and a second protein.

[0121] 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.

[0122] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

[0123] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

[0124] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CISH.

[0125] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

[0126] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

[0127] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

[0128] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

[0129] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

[0130] In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

[0131] In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

[0132] In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

[0133] In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

[0134] In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

[0135] In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

[0136] In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

[0137] In some embodiments, the first protein is PD-1 and the second protein is CISH.

[0138] In some embodiments, the first protein is CISH and the second protein is PD-1.

[0139] In some embodiments, the first protein is PD-1 and the second protein is CBL-B.

[0140] In some embodiments, the first protein is CBL-B and the second protein is PD-1.

[0141] In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

[0142] In some embodiments, the first protein is TIGIT and the second protein is PD-1.

[0143] In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

[0144] In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

[0145] In some embodiments, the first protein is CTLA-4 and the second protein is CISH.

[0146] In some embodiments, the first protein is CISH and the second protein is CTLA-4.

[0147] In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

[0148] In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

[0149] In some embodiments, the first protein is LAG-3 and the second protein is CISH.

[0150] In some embodiments, the first protein is CISH and the second protein is LAG-3.

[0151] In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

[0152] In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

[0153] In some embodiments, the first protein is CISH and the second protein is CBL-B.

[0154] In some embodiments, the first protein is CBL-B and the second protein is CISH.

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

[0156] In some embodiments, the first protein or the second protein is CTLA-4.

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

[0158] In some embodiments, the first protein or the second protein is CISH.

[0159] In some embodiments, the first protein or the second protein is CBL-B.

[0160] In some embodiments, the first protein or the second protein is TIGIT.

[0161] A.PD-1 One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 superfamily of T cell regulatory factors. Its ligands, PD-L1 and PD-L2, are expressed on a variety of tumor cells, including melanoma. The interaction of PD-1 with PD-L1 inhibits T cell effector function, leads to T cell exhaustion in the setting of chronic stimulation, and induces T cell apoptosis in the tumor microenvironment. PD-1 may also play a role in tumor-specific escape from immune surveillance.

[0162] According to certain embodiments, the expression of PD-1 in TILs is silenced or reduced according to the compositions and methods of the present invention. 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, and the method includes gene editing at least a portion of the TILs by silencing or suppressing the expression of PD-1. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as PD-1. For example, TALEN technology can be used to silence or reduce the expression of PD-1 in TILs.

[0163] B.CTLA-4 CTLA-4 expression is induced on activated T cells during T cell activation and competes for binding with antigen-presenting cell activation antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 causes T cell inhibition and helps maintain the balance of the immune response. However, inhibition of the interaction of CTLA-4 with CD80 or CD86 can prolong T cell activation and thus increase the level of immune response to cancer antigens.

[0164] According to certain embodiments, the expression of CTLA-4 in TILs is silenced or reduced according to the compositions and methods of the present invention. According to certain embodiments, the expression of both PD-1 and CTLA-4 in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), which method includes gene editing at least a portion of the TILs by silencing or suppressing the expression of CTLA-4. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as CTLA-4. For example, CRISPR, TALE, or zinc finger techniques can be used to silence or suppress the expression of CTLA-4 in TILs. In some embodiments, TALEN technology can be used to silence or reduce the expression of PD-1 and CTLA-4 in TILs.

[0165] C.LAG-3 Lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells after major histocompatibility complex (MHC) class II ligation. Although its mechanism remains unclear, its regulation causes a negative regulatory effect on T cell function, preventing tissue damage and autoimmunity. Therefore, LAG-3 blockade may improve antitumor responses. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

[0166] According to certain embodiments, the expression of LAG-3 in TILs is silenced or reduced according to the compositions and methods of the present invention. According to certain embodiments, the expression of both PD-1 and LAG-3 in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), which method includes gene editing at least a portion of the TILs by silencing or suppressing the expression of LAG-3. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as LAG-3. According to certain embodiments, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of LAG-3 in TILs. In some embodiments, TALEN technology can be used to silence or reduce the expression of PD-1 and LAG-3 in TILs.

[0167] D.Cish Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional inactivation against tumors. Genetic deletion of Cish in CD8+ T cells can enhance their expansion, functional affinity, and cytokine multifunction, resulting in significant and durable regression of established tumors. See, e.g., Palmer et al., Journal of Experimental Medicine, 212(12):2095 (2015).

[0168] According to certain embodiments, the expression of Cish in TILs is silenced or reduced according to the compositions and methods of the present invention. According to certain embodiments, the expression of both PD-1 and Cish in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), which method includes gene editing at least a portion of the TILs by silencing or suppressing the expression of Cish. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as Cish. For example, CRISPR, TALE, or zinc finger techniques can be used to silence or suppress the expression of Cish in TILs. In some embodiments, TALEN techniques can be used to silence or reduce the expression of PD-1 and Cish in TILs.

[0169] E.CBL-B CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and a negative regulator of T cell activation. Bachmaier, et al., Nature, 2000, 403, 211-216; Wallner, et al., Clin. Dev. Immunol. 2012, 692-639.

[0170] According to certain embodiments, expression of CBL-B in TILs is silenced or reduced according to the compositions and methods of the present invention. According to certain embodiments, expression of both PD-1 and CBL-B in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), which method includes gene editing at least a portion of the TILs by silencing or suppressing expression of CBL-B. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as CBL-B. For example, CRISPR, TALE, or zinc finger techniques can be used to silence or suppress expression of PKA in TILs. In some embodiments, CBL-B is silenced using TALEN knockout. In some embodiments, CBL-B is silenced using TALE-KRAB transcription inhibitor knock-in. Further details of these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some embodiments, TALEN methods can be used to silence or reduce the expression of PD-1 and CBL-B in TILs.

[0171] F.TIGIT 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) family of immunoglobulin proteins and suppresses T cell activation (Yu et al., Nat Immunol., 2009, 10(1):48-57). According to certain embodiments, the expression of TIGIT in TILs is silenced or reduced according to the compositions and methods of the present invention. According to certain embodiments, the expression of both PD-1 and TIGIT in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), which 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 can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as TIGIT. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of PKA in TILs. In some embodiments, TIGIT is silenced using TALEN knockout. In some embodiments, TIGIT is silenced using TALE-KRAB transcription inhibitor knock-in. Further details of these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585. In some embodiments, TALEN methods can be used to silence or reduce the expression of PD-1 and TIGIT in TILs.

[0172] IV. Gene Editing Methods As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that are genetically modified through gene editing to enhance their therapeutic effects. Embodiments of the present invention encompass gene editing via nucleotide insertion (RNA or DNA) into a TIL population to inhibit the expression of one or more proteins. Embodiments of the present invention also provide a method for expanding TILs into a therapeutic population, which method includes genetically editing the TILs. There are several gene editing techniques that can be used to genetically modify a TIL population, and they are suitable for use in the present invention.

[0173] In some embodiments, the method of genetically modifying a TIL population comprises the step of stable integration of a gene for the production of one or more proteins. In certain embodiments, the method of genetically modifying a TIL population comprises the step of retroviral transduction. In certain embodiments, the method of genetically modifying a TIL population comprises the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine, et al., Proc. 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 certain embodiments, the method of genetically modifying a TIL population comprises 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, a method for genetically modifying a TIL population includes a transposon-mediated gene transfer step. 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, thereby preventing long-term expression of the transposase, e.g., provided as mRNA (e.g., mRNA comprising a cap and polyA tail), in the transgenic cells. Suitable transposon-mediated gene transfer systems, including salmonid-type Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, as well as 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.

[0174] In some embodiments, the method for genetically modifying a TIL population includes the step of stable integration of a gene for the production or inhibition (e.g., silencing) of one or more proteins. In some embodiments, the method for genetically modifying a TIL population 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 each of which are incorporated herein by reference. Other electroporation methods known in the art can 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 are incorporated herein by reference). In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulse electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a defined and controlled permanent or temporary alteration of the TIL, and comprising applying to the TIL at least three single-operator-controlled, independently programmed DC electric pulse trains having a field strength of 100 V / cm or greater, wherein the at least three DC electric pulse trains have one, two, or three of the following characteristics: (1) at least two of the at least three pulses have pulse amplitudes that are different from each other; (2) at least two of the at least three pulses have pulse widths that are different from each other; and (3) the first pulse interval of a first set of two of the at least three pulses is different from the second pulse interval of a second set of two of the at least three pulses.In one embodiment, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a controlled, permanent or temporary alteration of the TIL's definition, comprising applying to the TIL at least three independently programmed DC electrical pulse trains controlled by a single operator having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses have different pulse amplitudes. In one embodiment, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a controlled, permanent or temporary alteration of the TIL's definition, comprising applying to the TIL at least three independently programmed DC electrical pulse trains controlled by a single operator having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses have different pulse widths. In one embodiment, the electroporation method is a pulsed electroporation method comprising treating TILs with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary alterations of the TILs, and comprising applying to the TILs at least three single-operator-controlled, independently programmed DC electrical pulse trains having a field strength of 100 V / cm or greater, wherein a first pulse interval of at least two of the at least three pulses is different from a second pulse interval of two of the at least three pulses.In one embodiment, the electroporation method is a pulsed electroporation method comprising treating TILs with a pulsed electric field to induce pore formation in the TILs, the method comprising applying to the TILs a train of at least three DC electric pulses having a field strength of 100 V / cm or greater, wherein the train 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 have pulse amplitudes that are different from each other; (2) at least two of the at least three pulses have pulse widths that are different from each other; and (3) a first pulse interval of a first set of two of the at least three pulses is different from a second pulse interval of a second set of two of the at least three pulses, thereby sustaining the induced pores for a relatively long period of time and maintaining the viability of the TILs. In one embodiment, the method for genetically modifying a TIL population comprises a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752, and U.S. Patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In certain embodiments, a method for genetically modifying a TIL population includes a liposomal transfection step.Liposomal transfection methods, such as those using a 1:1 (w / w) liposomal formulation of the cationic lipids N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphotidylethanolamine (DOPE), are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al. al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and 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 comprises a transfection step 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.

[0175] According to certain embodiments, the gene editing process can involve the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on the recognition of specific DNA sequences within the genome to target the nuclease domain to this position and mediate the generation of double-stranded breaks at the target sequence. The double-stranded break in DNA then recruits endogenous repair mechanisms to the break site to mediate genome editing by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Therefore, repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, suppress, or enhance) the target gene product.

[0176] The major classes of nucleases being 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). These nuclease systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs and TALENs achieve specific DNA binding through 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, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

[0177] Non-limiting examples of gene editing methods that can be used according to the TIL expansion method of the present invention include CRISPR, TALE, and ZFN methods, which are described in more detail below. According to certain embodiments, the method for expanding TILs into a therapeutic population can be carried out according to any embodiment of the method described herein (e.g., Process 2A) or as described in WO2018 / 081473A1, WO2018 / 129332A1, or WO2018 / 182817A1, and the method further includes gene editing at least a portion of the TILs using one or more of CRISPR, TALE, or ZFN methods to generate TILs that can provide an enhanced therapeutic effect. According to certain embodiments, the gene-edited TILs can be evaluated for improved therapeutic effect by comparing them with unmodified TILs in vitro, for example, by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs.

[0178] In some embodiments of the present invention, electroporation is used to deliver gene editing systems such as CRISPR systems, TALEN systems, 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 suitable for use with some embodiments of the present invention is the commercially available MaxCyte STX system. There are several alternative commercially available electroporation devices that may be suitable for use with the present invention, such as the AgilePulse system or ECM830 available from BTX-Harvard Apparatus, 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, together with the remainder of the TIL expansion method, forms a sterile, closed system. In some embodiments of the present invention, the electroporation system is a pulse electroporation system as described herein, and, together with the remainder of the TIL expansion method, forms a sterile, closed system.

[0179] A.TALE Method The method for expanding TILs into a therapeutic population can be carried out according to any of the embodiments of the methods described herein, or as described in WO2018 / 081473A1, WO2018 / 129332A1, or WO2018 / 182817A1, wherein the method further comprises gene editing at least a portion of the TILs using the TALE method. According to certain embodiments, the use of the TALE method during the TIL expansion process silences or reduces the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the TALE method during the TIL expansion process enhances the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

[0180] TALE is an abbreviation for "Transcription Activator-Like Effector" proteins, including TALENs ("Transcription Activator-Like Effector Nucleases"). Methods 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 pathogenic fungus Xanthomonas genus. They contain a DNA-binding domain composed of a series of 33-35 amino acid repeat domains, each recognizing a single base pair. TALE specificity is determined by two hypervariable amino acids known as repeat variable dinucleotides (RVDs). Modular TALE repeat sequences are joined together to recognize adjacent DNA sequences. Specific RVDs within the DNA-binding domain recognize bases within the target locus and provide structural features for assembling predictable DNA-binding domains. The DNA-binding domain of TALEs is fused to the catalytic domain of a type IIS FokI endonuclease to create targetable TALE nucleases. To induce site-specific mutations, two individual TALEN arms, separated by a 14–20 base pair spacer region, bring FokI monomers into close proximity and dimerize, producing the targeted double-stranded break.

[0181] Several large-scale systematic studies utilizing various assembly methods have shown that TALE repeats can be combined to recognize virtually 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). TALE and TALEN methods suitable for use in the present invention are described in U.S. Patent Publication Nos. US2011 / 0201118A1, US2013 / 0117869A1, US2013 / 0315884A1, US2015 / 0203871A1, and US2016 / 0120906A1, the disclosures of which are incorporated herein by reference.

[0182] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing TILs using 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, TNFRSF 10B, 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.

[0183] Non-limiting examples of TALE nucleases targeting the PD-1 gene are provided in the table below. In these examples, the targeted genomic sequence comprises two 17-base pair (bp) long sequences (shown in uppercase letters and referred to as half targets) separated by a 15-bp spacer (shown in lowercase letters). Each half target is recognized by a repeat of the half TALE nuclease listed in the table. Thus, according to certain embodiments, the TALE nuclease according to the present invention recognizes and cleaves a target sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14. TALEN sequences and gene editing methods are also described in Gautron et al., Molecular Therapy: Nucleic Acids Dec. 2017, Vol. 9: 312-321, which is incorporated herein by reference.

[0184] [Table 3]

[0185] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10]

[0186] Non-limiting examples of genes that can be enhanced by permanently gene editing TILs via the TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15, and IL-21.

[0187] Examples of systems, methods, and compositions that can be used in accordance with embodiments of the present invention for altering expression of target gene sequences by the TALE method are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference.

[0188] B.2.Cas-CLOVER method The method for expanding TILs into a therapeutic population can be carried out according to any embodiment of the method described herein (e.g., Process 2A), or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene editing at least a portion of the TILs using the Cas-CLOVER method. According to certain embodiments, the use of the Cas-CLOVER method during the TIL expansion process silences or reduces the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the CLOVER method during the TIL expansion process enhances the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

[0189] Cas-CLOVER is a dimeric, high-fidelity site-specific nuclease (SSN) consisting of a fusion of catalytically inactive SpCas9 (dCas9) with the nuclease domain of the Clostridial CloO51 type IIs restriction endonuclease (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 is predicted based on the dimerization of the CloO51 nuclease domain, enabled by RNA-guided recognition of two adjacent 20-nt target sequences. Unlike paired nickase approaches, for example, 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.

[0190] 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 comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule.

[0191] DNA localization component In embodiments, the DNA localization component can bind to a specific DNA sequence. In embodiments, the DNA localization component is selected from, for example, a DNA-binding oligonucleotide, a DNA-binding protein, a DNA-binding protein complex, and combinations thereof. Other suitable DNA-binding components will be recognized by those skilled in the art.

[0192] In embodiments, the DNA localization component comprises an oligonucleotide directed to a specific locus(s) within the genome. The oligonucleotide may be selected from DNA, RNA, DNA / RNA hybrids, and combinations thereof.

[0193] In embodiments, the DNA localization component comprises a nucleotide-binding protein or protein complex that binds to the 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 localization component comprises a protein or protein complex capable of recognizing an R-loop selected from Cas9, the Cascade complex, RecA, RNase H, an RNA polymerase, a DNA polymerase, or a combination thereof. In embodiments, the DNA localization component comprises an engineered protein capable of binding to the target DNA. In embodiments, the DNA localization component comprises a protein capable of binding to a DNA sequence selected from a meganuclease, a zinc finger array, a transcription activator-like (TAL) array, and combinations thereof. In embodiments, the DNA localization component comprises a protein comprising a naturally occurring DNA-binding domain. In embodiments, the DNA localization 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 localization component comprises an oligonucleotide directed to a specific locus within a genome. Exemplary oligonucleotides include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and any combination thereof. In embodiments, the DNA localization component comprises a protein or protein complex capable of recognizing a feature selected from an RNA-DNA heteroduplex, an R-loop, and any combination thereof. Exemplary proteins or protein complexes 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 capable of recognizing an R-loop comprises Cas9.In embodiments, the DNA localization component comprises a protein capable of binding to a DNA sequence selected from a meganuclease, a zinc finger array, a TAL array, and any combination thereof, hi embodiments, the DNA localization component comprises an oligonucleotide directed to a target location within a genome and a protein capable of binding to the target DNA sequence.

[0194] In embodiments, the DNA localization component comprises, consists essentially of, or consists of at least one guide RNA (gRNA). In embodiments, the DNA localization component comprises, consists essentially of, or consists of two gRNAs, where 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 localization 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). In embodiments, the DNA localization component comprises, consists essentially of, or consists of the DNA binding domain of a TALEN or TAL protein derived from Xanthomonas or Ralstonia.

[0195] Effector molecules In embodiments, the effector molecule can exert a predetermined effect at a specific locus within 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.

[0196] In embodiments, the effector molecule comprises a transposase. In embodiments, the effector molecule comprises a PB transposase (PBase). In embodiments, the effector molecule comprises a nuclease. Non-limiting examples of nucleases include restriction endonucleases, homing endonucleases, S1 nuclease, mungbean nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO endonuclease, or any combination thereof. In certain embodiments, the effector molecule comprises a restriction endonuclease. In certain embodiments, the effector molecule comprises a type IIS restriction endonuclease. In embodiments, the effector molecule comprises 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, BpulIOI, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, and Clo051. In embodiments, the effector molecule comprises BmrI, BfiI, or Clo051.

[0197] In embodiments, the effector molecule comprises, consists essentially of, or consists of a homodimer or heterodimer. In embodiments, the effector molecule comprises, consists essentially of, or consists of a nuclease, optionally an endonuclease. In embodiments, effector molecules, including those homodimer- or heterodimer-containing effector molecules, comprise, consist essentially of, or consist of Cas9, a Cas9 nuclease domain, or a fragment thereof. In embodiments, the Cas9 is a catalytically inactive or "deactivated" Cas9 (dCas9 (SEQ ID NOs: 302 and 303 of WO2019 / 126578)). In embodiments, the Cas9 is a catalytically inactive or "deactivated" nuclease domain of Cas9. In embodiments, dCas9 is encoded by a shorter sequence derived from a full-length, catalytically inactivated Cas9, referred to herein as "small" dCas9 or dSaCas9 (SEQ ID NO: 23 of WO2019 / 126578).

[0198] In fusion protein embodiments, the effector molecule comprises, consists essentially of, or consists of one or more homodimers or heterodimers of type II nucleases. In fusion protein embodiments, the effector molecule comprises, consists essentially of, or consists of homodimers or heterodimers of type II nucleases. In embodiments, the type II nuclease comprises 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, BpulIOI, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, or Clo051.

[0199] In embodiments, effector molecules, including those containing homodimers or heterodimers, comprise, consist essentially of, or consist of Clo051, BfiI, or BmrI. In embodiments, effector molecules, including those containing homodimers or heterodimers, comprise, consist essentially of, or consist of Cas9, a Cas9 nuclease domain, or a fragment thereof that forms a heterodimer with Clo051, BfiI, or BmrI. In embodiments, effector molecules, including those containing homodimers or heterodimers, comprise, consist essentially of, 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 Clo051 nuclease domain may comprise, consist essentially of, or consist of the following amino acid sequence: EGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRH LGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVN PNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAE KIRSGEMTIEELERAMFNNSEFILKY (SEQ ID NO: 23).

[0200] In embodiments, effector molecules, including homodimeric or heterodimeric effector molecules thereof, comprise, consist essentially of, or consist of the DNA-binding domain of a TALEN or TAL protein from Xanthomonas or Ralstonia. In embodiments, effector molecules, including homodimeric or heterodimeric effector molecules thereof, comprise, consist essentially of, or consist of the 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 homodimeric or heterodimeric effector molecules thereof, comprise, consist essentially of, or consist of the DNA-binding domain of a TALEN or TAL protein from Xanthomonas or Ralstonia that forms a homodimer or heterodimer with Clo051.

[0201] join In embodiments, a fusion protein comprises, consists essentially of, or consists of a DNA localization component and an effector molecule. In embodiments, nucleic acid sequences encoding one or more components of a 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 also includes a linker region operably linking the two components of the fusion protein. For example, in embodiments, the fusion protein comprises, consists essentially of, or consists of a DNA localization component and an effector molecule operably linked by the linker region. In embodiments, the DNA localization 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 results in a fusion protein. In embodiments, the fusion protein can include a non-covalent linkage between the DNA localization component and the effector molecule. The non-covalent linkage can include an antibody, antibody fragment, antibody mimetic, or scaffold protein.

[0202] fusion proteins In embodiments, the DNA localization component comprises, consists essentially of, or consists of at least one gRNA, and the effector molecule comprises, consists essentially of, or consists of Cas9, a Cas9 nuclease domain, or a fragment thereof. In embodiments, the DNA localization component comprises, consists essentially of, or consists of at least one gRNA, and the effector molecule comprises, consists essentially of, or consists of an inactivated Cas9 (dCas9) or an inactivated nuclease domain. In embodiments, the DNA localization component comprises, consists essentially of, or consists of at least one gRNA, and the effector molecule comprises, consists essentially of, or consists of an inactivated small Cas9 (dSaCas9). In embodiments, the effector molecule comprises, consists essentially of, or consists of Cas9, dCas9, dSaCas9, or a nuclease domain thereof, and a second endonuclease. The second endonuclease is AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, M bo1I, 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.

[0203] In embodiments of the fusion protein, the DNA localization 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 protein of the present disclosure, the DNA localization component comprises, consists essentially of, or consists of the DNA binding domain of a TALEN or TAL protein from Xanthomonas or Ralstonia, and the effector molecule comprises, consists essentially of, or consists of an endonuclease. or Clo051.

[0204] In certain embodiments, an exemplary dCas9-Clo051 fusion protein can comprise, consist essentially of, or consist of 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.

[0205] construct In embodiments, the nuclease domain comprises, consists essentially of, or consists of dCas9 and Clo051. In embodiments, the nuclease domain comprises, consists essentially of, or consists of dSaCas9 and Clo051. In embodiments, the nuclease domain comprises, consists essentially of, or consists of Xanthomonas-TALE and Clo051. In embodiments, the nuclease domain comprises, consists essentially of, or consists of Ralstonia-TALE and Clo051. In embodiments, the fusion protein comprises dCas9-Clo051, dSaCas9-Clo051, Xanthomonas-TALE-Clo051, or Ralstonia-TALE-Clo051. In embodiments, the vector encoding the fusion protein comprises Csy4-T2A-Clo051-G4S linker-dCas9 (Streptoccocus pyogenes) or pRT1-Clo051-dCas9 double NLS.

[0206] According to some embodiments, the Cas-CLOVER system comprises a fusion protein comprising a DNA-localizing component and an effector molecule, wherein the DNA-localizing component hybridizes to a target sequence of a DNA molecule in the TIL, the DNA molecule encodes at least one immune checkpoint molecule, the TIL expresses the at least one immune checkpoint molecule, and the effector molecule cleaves the DNA molecule, thereby altering expression of the at least one immune checkpoint molecule.

[0207] According to certain embodiments, the Cas-CLOVER method involves 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) specific to the target DNA sequence of the immune checkpoint gene(s). The fusion protein can be delivered as DNA, mRNA, or protein. Upon contact of the genome with the Cas-CLOVER system, one or more strands of the target double-stranded DNA can be cleaved. If the cleavage occurs in the presence of one or more DNA repair pathways or their components, the Cas-CLOVER method either disrupts gene expression or modifies the genomic sequence by inserting, deleting, or substituting one or more base pairs. DSBs can be repaired intracellularly by non-homologous end joining (NHEJ), a mechanism that frequently causes DNA insertions or deletions (indels). Indels often result in a frameshift, creating a loss of function allele, for example, by causing a premature stop codon within the open reading frame (ORF) of the target gene. According to certain embodiments, the result is a loss-of-function mutation within the target immune checkpoint gene.

[0208] Alternatively, DSBs induced by the Cas-CLOVER system can be repaired by homology-directed repair (HDR) instead of NHEJ. NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, but homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions. According to some embodiments, HDR is used to gene-edit immune checkpoint genes by delivering a DNA repair template containing the desired sequence to TILs using the Cas-CLOVER system. The repair template preferably contains the desired edit as well as additional homologous sequences (often referred to as left and right homologous arms) immediately upstream and downstream of the target gene.

[0209] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing TILs 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, T These include NFRSF10A, 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.

[0210] Examples of systems, methods, and compositions that can be used in accordance with embodiments of the present invention for altering expression of target gene sequences via the Cas-CLOVER method 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 entireties. Resources for implementing the Cas-CLOVER method, such as CLOVER mRNA and Cas-CLOVER mRNA constructs, are commercially available from companies such as Demeetra and Hera Biolabs.

[0211] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second TIL population to transfer at least one gene editor into a plurality of cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion 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) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) optionally, cryopreserving the harvested TIL population using a cryopreservation medium; The electroporation step includes delivery of at least one gene editor system comprising a Cas-CLOVER system, wherein the at least one gene editor system regulates expression of at least one checkpoint protein in a plurality of cells of the second TIL population.

[0212] According to some embodiments, a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (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) occurs without opening the system; and (e) sterile electroporating the second TIL population to transfer at least one gene editor into a plurality of cells of the second TIL population; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion 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) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) optionally, cryopreserving the harvested TIL population using a cryopreservation medium; The electroporation step includes delivery of at least one gene editor system comprising the Cas-CLOVER system, wherein the at least one gene editor system inhibits expression of at least one checkpoint protein in a plurality of cells of the second TIL population.

[0213] V. Methods of Using Gene-Edited TILs to Treat Cancer Patients Some embodiments disclosed herein provide a method for treating a cancer patient, the method comprising: (a) generating a gene-edited TIL population from tumors resected from cancer patients; and (b) assessing the proliferation index of the gene-edited TIL population using the methods disclosed herein; and (c) administering a therapeutically effective dose of gene-edited TILs to the cancer patient if the proliferation index of the gene-edited TIL population is lower than the proliferation index of the reference value.

[0214] The proliferation level of gene-edited T cells in the absence of IL-2 can be expressed as a number, such as a proliferation index (PI), which reflects the transformant state of the gene-edited T cells according to the methods of the present invention. This proliferation level of gene-edited T cells in the absence of IL-2 can be compared, for example, with the proliferation level of a positive control sample of a transformed cell line (e.g., Jurkat cells) that exhibits IL-2-independent growth, the proliferation level of a positive control sample of gene-edited T cells in the presence of IL-2, or both. Furthermore, the proliferation level of gene-edited T cells in the absence of IL-2 can be compared, for example, 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 these pieces of information, the proliferation index (PI) according to the following formula can be used as an assessment measure of the transformation state of the gene-edited T cells: PI = (proliferation level of gene-edited T cells in the absence of IL-2), PI = (proliferation level of gene-edited T cells in the absence of IL-2) - (proliferation level of negative control sample in the absence of IL-2), PI = (proliferation level of gene-edited T cells in the absence of IL-2) / (proliferation level of positive control sample in the absence of IL-2), or PI = [(proliferation level of gene-edited T cells in the absence of IL-2) - (proliferation level of negative control sample in the absence of IL-2)] / (proliferation level of positive control sample in the absence of IL-2).

[0215] In some embodiments, the reference value may be the proliferation index of the positive control. In some embodiments, the reference value may be 90% of the proliferation index of the positive control. In some embodiments, the reference value may be 80% of the proliferation index of the positive control. In some embodiments, the reference value may be 70% of the proliferation index of the positive control. In some embodiments, the reference value may be 60% of the proliferation index of the positive control. In some embodiments, the reference value may be 50% of the proliferation index of the positive control. In some embodiments, the reference value may be 40% of the proliferation index of the positive control. In some embodiments, the reference value may be 30% of the proliferation index of the positive control. In some embodiments, the reference value may be 20% of the proliferation index of the positive control. In some embodiments, the reference value may be 10% of the proliferation index of the positive control. In some embodiments, the reference value may be 5% of the proliferation index of the positive control.

[0216] In some embodiments, the reference value can be the proliferation index of the negative control. In some embodiments, the reference value can be the proliferation index of the negative control + / - 2StdDev.

[0217] In some embodiments, the reference value can be about 0 to about 0.9. In some embodiments, the reference value can be about 0 to about 0.8. In some embodiments, the reference value can be about 0 to about 0.7. In some embodiments, the reference value can be about 0 to about 0.9. In some embodiments, the reference value can be about 0 to about 0.6. In some embodiments, the reference value can be about 0 to about 0.5. In some embodiments, the reference value can be about 0 to about 0.4. In some embodiments, the reference value can be about 0 to about 0.3. In some embodiments, the reference value can be about 0 to about 0.2. In some embodiments, the reference value can be about 0 to about 0.1. In some embodiments, the reference value can be 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 may be about 0.4. In some embodiments, the reference value may be about 0.3. In some embodiments, the reference value may be about 0.2. In some embodiments, the reference value may be about 0.1. In some embodiments, the reference value may be about 0.05.

[0218] The compositions and methods described herein can be used in methods for treating diseases. In some embodiments, the compositions and methods described herein are for use in treating hyperproliferative disorders, such as cancer, in adult or pediatric patients. The compositions and methods described herein can also be used to treat other disorders described herein and in the following paragraphs.

[0219] 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 cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma, and primitive neuroectogerminal tumor), cervical cancer (including squamous cell cervical carcinoma, adenosquamous cervical carcinoma, and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophagogastric junction cancer, gastric cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypopharyngeal cancer, and esophageal and rectal cancers). 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's sarcoma, osteosarcoma, rhabdomyosarcoma, and other sarcomas of bone and soft tissue), thyroid cancer (including anaplastic thyroid carcinoma), uterine cancer, and vaginal cancer.

[0220] In some embodiments, the hyperproliferative disorder is a hematological malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some embodiments, the invention includes methods of treating a patient with cancer, wherein the cancer is a hematological malignancy. In some embodiments, the invention includes methods of treating a patient with cancer using TILs, MILs, or PBLs 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 invention includes methods of treating patients with cancer using MILs or PBLs modified to downregulate one or more of PD-1, CTLA-4, LAG-3, CISH, and CBL-BR, wherein the cancer is a hematological malignancy.

[0221] In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, that has relapsed or is refractory to treatment with at least one previous therapy, including chemotherapy, radiation therapy, or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers that has relapsed or is refractory to treatment with at least two previous therapies, including chemotherapy, radiation therapy, and / or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers that has relapsed or is refractory to treatment with at least three previous therapies, including chemotherapy, radiation therapy, and / or immunotherapy.

[0222] In some embodiments, the cancer is a microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) cancer. Accordingly, MSI-H and dMMR cancers and tests are described in Kawakami, et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosure of which is incorporated herein by reference.

[0223] In some embodiments, the invention includes methods of treating a patient having cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor and / or a MEK inhibitor. In some embodiments, the invention includes methods of treating a patient having cancer, wherein the cancer is refractory to treatment 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 invention includes methods of treating a patient having cancer, wherein the cancer is refractory to treatment 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 refractory to treatment with a BRAF inhibitor selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof, and a MEK inhibitor selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof.

[0224] In some embodiments, the present invention includes a method of treating a patient with cancer, wherein the cancer is a childhood cancer.

[0225] In some embodiments, the present invention includes a method of treating a patient with cancer, wherein the cancer is uveal melanoma.

[0226] In some embodiments, the invention includes a method of treating a patient with cancer, wherein the uveal melanoma is choroidal melanoma, ciliary body melanoma, or iris melanoma.

[0227] In some embodiments, the invention includes a method of treating a patient with cancer, wherein the pediatric cancer is neuroblastoma.

[0228] In some embodiments, the invention includes a method of treating a patient with cancer, wherein the pediatric cancer is a sarcoma.

[0229] In some embodiments, the present invention includes a method of treating a patient with cancer, wherein the sarcoma is osteosarcoma.

[0230] In some embodiments, the present invention includes a method of treating a patient with cancer, wherein the sarcoma is a soft tissue sarcoma.

[0231] In some embodiments, the invention includes a method of treating a patient with cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

[0232] In some embodiments, the present invention includes a method of treating a patient with cancer, wherein the pediatric cancer is a central nervous system (CNS)-related cancer. In some embodiments, the pediatric cancer is refractory to treatment with chemotherapy. In some embodiments, the pediatric cancer is refractory to treatment with radiation therapy. In some embodiments, the pediatric cancer is refractory to treatment with dinutuximab.

[0233] In some embodiments, the invention includes a method of treating a patient with cancer, wherein the CNS-related cancer is medulloblastoma, pineoblastoma, glioma, ependymoma, or glioblastoma.

[0234] The compositions and methods described herein may be used in methods for treating cancer, wherein the cancer is refractory or resistant to prior treatment with an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient is a primary refractory patient to an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient has no prior response to an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient has a prior response to an anti-PD-1 or anti-PD-L1 antibody, followed by progression of the patient's cancer. In some embodiments, the cancer is refractory to an anti-CTLA-4 antibody and / or an anti-PD-1 or anti-PD-L1 antibody in combination with at least one chemotherapeutic agent. In some embodiments, the prior chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and / or cisplatin. In some prior embodiments, the chemotherapeutic agent(s) is a platinum-doublet chemotherapeutic agent. In some embodiments, the platinum doublet therapy comprises a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and taxanes (including, for example, paclitaxel, docetaxel, or nab-paclitaxel). In some embodiments, the platinum doublet chemotherapeutic agent is combined with pemetrexed.

[0235] In some embodiments, the NSCLC is from a patient with a cancer that is PD-L1 negative and / or expresses PD-L1 with a tumor proportion score (TPS) of <1%, as described elsewhere herein.

[0236] In some embodiments, the NSCLC is refractory to a combination therapy comprising anti-PD-1 or anti-PD-L1 and platinum doublet therapy, wherein the platinum doublet therapy is i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin; ii) a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and a taxane (including, for example, paclitaxel, docetaxel, or nab-paclitaxel).

[0237] In some embodiments, the NSCLC is refractory to a combination therapy comprising an anti-PD-1 or anti-PD-L1 antibody, pemetrexed, and platinum doublet therapy, wherein the platinum doublet therapy is i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin; ii) a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and a taxane (including, for example, paclitaxel, docetaxel, or nab-paclitaxel).

[0238] In some embodiments, the NSCLC has been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC patient is treatment-naive. In some embodiments, the NSCLC has not been treated with an anti-PD-1 antibody. In some embodiments, the NSCLC has not been treated with an anti-PD-L1 antibody. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent but is no longer being treated with the chemotherapeutic agent. In some embodiments, the NSCLC patient is anti-PD-1 / PD-L1 naive. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the NSCLC patient has treatment-naive NSCLC or is post-chemotherapeutic treatment but is anti-PD-1 / PD-L1 naive. In some embodiments, the NSCLC patient is treatment-naive or is post-chemotherapeutic treatment but is anti-PD-1 / PD-L1 naive and has low PD-L1 expression. In some embodiments, the NSCLC patient has bulky mass disease at baseline. In some embodiments, the subject has bulky mass disease at baseline and low PD-L1 expression. In some embodiments, the NSCLC patient has no detectable PD-L1 expression. In some embodiments, the NSCLC patient is treatment-naive or post-chemotherapy treatment but is anti-PD-1 / PD-L1 naive and has no detectable PD-L1 expression. In some embodiments, the patient has bulky mass disease at baseline and no detectable PD-L1 expression. In some embodiments, the NSCLC patient has treatment-naive NSCLC or post-chemotherapy (e.g., post-chemotherapeutic agent) but is anti-PD-1 / PD-L1 naive with low PD-L1 expression and / or bulky mass disease at baseline. In some embodiments, bulky mass disease is indicated when the maximum tumor diameter is greater than 7 cm measured in either the transverse or coronal plane. In some embodiments, bulky mass disease is indicated when there are enlarged lymph nodes with a short-axis diameter of 20 mm or greater.In some embodiments, the chemotherapeutic agent comprises a standard of care therapeutic agent for NSCLC.

[0239] In some embodiments, PD-L1 expression is determined by a tumor proportion score. In some embodiments, the subject with refractory NSCLC tumors has a tumor proportion score (TPS) of <1%. In some embodiments, the subject with refractory NSCLC tumors has a TPS of ≥1%. In some embodiments, the 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 the anti-PD-1 and / or anti-PD-L1 antibody treatment. In some embodiments, the subject with refractory NSCLC has been previously treated with an anti-PD-L1 antibody, and the tumor proportion score was determined prior to the anti-PD-L1 antibody treatment.

[0240] In some embodiments, PD-L1 expression is determined by a tumor proportion score using one of the additional testing methods described herein. In some embodiments, the 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, the subject or patient with NSCLC has previously been treated with an anti-PD-1 and / or anti-PD-L1 antibody, and the tumor proportion score was determined prior to anti-PD-1 and / or anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with NSCLC has previously been treated with an anti-PD-L1 antibody, and the tumor proportion score was determined prior to anti-PD-L1 antibody treatment. In some embodiments, the subject or patient with refractory or resistant NSCLC tumor has a tumor proportion score (TPS) of <1%. In some embodiments, the subject or patient with refractory or resistant NSCLC tumor has a TPS of ≥1%. In some embodiments, the 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 anti-PD-1 and / or anti-PD-L1 antibody treatment. In some embodiments, the 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 anti-PD-L1 antibody treatment.

[0241] In some embodiments, the NSCLC is an NSCLC from a patient taken prior to anti-PD-1 or anti-PD-L1 therapy that exhibits partial or complete membrane staining of any intensity for PD-L1 protein of less than 1% (TPS<1%). In some embodiments, the 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 of 0% to 1%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.9%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.8%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.7%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.6%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.5%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.4%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.3%.In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.2%. In some embodiments, the NSCLC is an NSCLC exhibiting a TPS of 0% to 0.1%. TPS can be measured by methods known in the art, such as the method described in Hirsch, et al. J. Thorac. Oncol. 2017, 12, 208-222, or by methods used to determine TPS before treatment with pembrolizumab or other anti-PD-1 or anti-PD-L1 therapy. Methods approved by the US Food and Drug Administration for measuring TPS can also be used. In some embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

[0242] 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% membrane staining.

[0243] In some embodiments, testing for PD-L1 may involve measuring the level of PD-L1 in the patient's serum. In these embodiments, measuring PD-L1 in the patient's serum eliminates the uncertainty of tumor heterogeneity and the patient's discomfort with serial biopsies.

[0244] In some embodiments, elevated soluble PD-L1 compared to baseline or standard levels correlates with worse prognosis in NSCLC. See, e.g., Okuma, et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli, et al., Oncotarget, 2018, 9, 17554-17563. In some embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is expressed on circulating tumor cells.

[0245] In some embodiments, the subject or patient has non-small cell lung cancer (NSCLC) characterized by at least one of the following: i. PD-L1 defined tumor proportion score (TPS) < 1%; ii. PD-L1 TPS score of 1% to 49%, or iii. the predetermined absence of one or more driver mutations; The driver mutation is selected from the group consisting of an EGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion, a RET mutation, a RET fusion, an ERBB2 mutation, an ERBB2 amplification, a BRCA mutation, a MAP2K1 mutation, a PIK3CA, a CDKN2A, a PTEN mutation, a UMD mutation, an NRAS mutation, a KRAS mutation, an NF1 mutation, a MET mutation, a MET splice and / or altered MET signaling, a TP53 mutation, a CREBBP mutation, a KMT2C mutation, a KMT2D mutation, an ARID1A mutation, an RB1 mutation, an ATM mutation, a SETD2 mutation, a FLT3 mutation, a PTPN11 mutation, an FGFR1 mutation, an EP300 mutation, a MYC mutation, an EZH2 mutation, a JAK2 mutation, a FBXW7 mutation, a CCND3 mutation, and a GNA11 mutation.

[0246] In other embodiments, the present invention provides methods for treating a subject with cancer, comprising administering to the subject a therapeutically effective dose of a therapeutic TIL population described herein.

[0247] In other embodiments, the present invention provides methods for treating a subject with cancer, comprising administering to the subject a therapeutically effective dose of a TIL composition described herein.

[0248] In other embodiments, the present invention provides methods for treating a subject having a cancer described herein, modified such that the subject is administered a non-myeloablative lymphodepletion regimen prior to administering a therapeutically effective dose of the therapeutic TIL populations and TIL compositions described herein, respectively.

[0249] In other embodiments, the invention provides a method for treating a subject having a cancer described herein, wherein the non-myeloablative lymphodepletion regimen is modified to include 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.

[0250] In other embodiments, the invention provides methods for treating a subject having cancer as described herein, modified to further include treating the subject with a high-dose IL-2 regimen starting the day after administering the TIL cells to the subject.

[0251] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the high-dose IL-2 regimen is modified to include 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0252] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be a solid tumor.

[0253] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, modified so 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 papillary carcinoma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

[0254] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, modified such that the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0255] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be melanoma.

[0256] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be metastatic melanoma.

[0257] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be HNSCC.

[0258] In other embodiments, the present invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be cervical cancer.

[0259] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be NSCLC.

[0260] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be metastatic NSCLC.

[0261] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be glioblastoma (including GBM).

[0262] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be a gastrointestinal cancer.

[0263] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer has been modified to be a hypermutated cancer.

[0264] In other embodiments, the invention provides methods for treating a subject having a cancer described herein, wherein the cancer is modified to be a childhood hypermutated cancer.

[0265] In other embodiments, the present invention provides a therapeutic TIL population as described herein for use in a method for treating a subject having cancer, comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

[0266] In other embodiments, the present invention provides a TIL composition described herein for use in a method for treating a subject having cancer, comprising administering a therapeutically effective dose of the TIL composition to the subject.

[0267] In other embodiments, the present invention provides a therapeutic TIL population described herein or a TIL composition described herein that has been modified such that a non-myeloablative lymphodepletion regimen has been administered to the subject prior to administering a therapeutically effective dose of the therapeutic TIL population described herein or the TIL composition described herein to the subject.

[0268] In another embodiment, the present invention provides a method for administering a non-myeloablative lymphodepletion regimen comprising administering a non-myeloablative lymphodepletion regimen to a patient in need thereof, the method comprising administering a non-myeloablative lymphodepletion regimen to a patient in need thereof, the non-myeloablative lymphodepletion regimen being administered in a dose of 60 mg / m 2 / day for 2 days followed by cyclophosphamide at a dose of 25 mg / m 2 The present invention provides a therapeutic TIL population or TIL composition as described herein, modified to include administering fludarabine at a dose of 100 mg / day for 5 days.

[0269] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein modified to further include a step of treating the patient with a high-dose IL-2 regimen starting the day after administration of the TIL cells to the patient.

[0270] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, wherein the high-dose IL-2 regimen is modified to include 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0271] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is a solid tumor.

[0272] In other embodiments, the invention provides a therapeutic TIL population or TIL composition as described herein modified so 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 papillary carcinoma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, or renal cell carcinoma.

[0273] In other embodiments, the present invention provides therapeutic TIL populations or TIL compositions described herein modified such that the cancer is melanoma, metastatic melanoma, HNSCC, cervical cancer, NSCLC, metastatic NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0274] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is melanoma.

[0275] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is metastatic melanoma.

[0276] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is HNSCC.

[0277] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is cervical cancer.

[0278] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, wherein the cancer is NSCLC.

[0279] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, wherein the cancer is metastatic NSCLC.

[0280] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is glioblastoma.

[0281] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is a gastrointestinal cancer.

[0282] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, wherein the cancer is modified to be a hypermutated cancer.

[0283] In other embodiments, the present invention provides a therapeutic TIL population or TIL composition described herein, modified such that the cancer is a pediatric hypermutated cancer.

[0284] In other embodiments, the present invention provides the use of a therapeutic TIL population described herein in a method of treating cancer in a subject, comprising administering a therapeutically effective dose of the therapeutic TIL population to the subject.

[0285] In other embodiments, the invention provides the use of a TIL composition described in any of the preceding paragraphs in a method for treating cancer in a subject, comprising administering a therapeutically effective dose of the TIL composition to the subject.

[0286] In other embodiments, the invention provides use of a therapeutic TIL population described herein or a TIL composition described herein in a method of treating cancer in a patient comprising administering to the patient a non-myeloablative lymphodepletion regimen and then administering to the subject a therapeutically effective dose of a therapeutic TIL population described in any of the preceding paragraphs or a therapeutically effective dose of a TIL composition described herein.

[0287] 1. Lymphocyte depletion preconditioning of patients In some embodiments, the invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the present disclosure. In some embodiments, the invention includes a TIL population for use in treating cancer in a patient who has been pretreated with non-myeloablative chemotherapy. In some embodiments, the TIL population is for administration by infusion. In some embodiments, the non-myeloablative chemotherapy includes cyclophosphamide 60 mg / kg / day for two days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m 2 / day for 5 days (27-23 days prior to TIL infusion). In some embodiments, after non-myeloablative chemotherapy according to the present disclosure and TIL infusion (day 0), the patient receives an intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) at 720,000 IU / kg intravenously every 8 hours to physiological tolerance. In certain embodiments, the TIL population is for use in treating cancer in combination with IL-2, and the IL-2 is administered after the TIL population.

[0288] Experimental results indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy 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 referred to as "immunosuppressive conditioning") in patients prior to introducing the TILs of the present invention.

[0289] Typically, lymphocyte depletion is achieved using the administration of fludarabine or cyclophosphamide (the active form is called 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.

[0290] In some embodiments, fludarabine is administered at a concentration of 0.5 μg / mL to 10 μg / mL of fludarabine. In some embodiments, fludarabine is administered at a concentration of 1 μg / mL of fludarabine. In some embodiments, fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, fludarabine is administered at a dose of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day. In some embodiments, fludarabine treatment is administered at 35 mg / kg / day for 2 to 7 days. In some embodiments, fludarabine treatment is administered at 35 mg / kg / day for 4 to 5 days. In some embodiments, fludarabine treatment is administered at 25 mg / kg / day for 4-5 days.

[0291] 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 treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, cyclophosphamide is administered at a concentration of 100 mg / m 2 / day, 150mg / m 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day, or 300 mg / m 2 In some embodiments, cyclophosphamide is administered at a dose of 35 mg / kg / day for 2-7 days. In some embodiments, cyclophosphamide is administered at a dose of 250 mg / m 2 / day for 4-5 days. In some embodiments, cyclophosphamide treatment is administered at a dose of 250 mg / m 2 / day for 4 days.

[0292] In some embodiments, lymphodepletion is performed by administering fludarabine and cyclophosphamide together to the patient. In some embodiments, fludarabine is administered at a dose of 25 mg / m 2 / day and cyclophosphamide at 250 mg / m 2 / day for 4 days.

[0293] In some embodiments, lymphodepletion is achieved by administering cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 This is performed by administering the drug at a dose of 100 mg / day for 5 days.

[0294] In some embodiments, lymphodepletion is achieved by administering cyclophosphamide at 60 mg / m 2 / day for 2 days, and fludarabine 25 mg / m 2 / day for 5 days, with cyclophosphamide and fludarabine both administered on the first 2 days, for a total of 5 days of lymphodepletion.

[0295] In some embodiments, lymphodepletion is achieved by administering cyclophosphamide at about 50 mg / m 2 / day for 2 days, with fludarabine at approximately 25 mg / m 2 / day for 5 days, with cyclophosphamide and fludarabine both administered on the first 2 days, for a total of 5 days of lymphodepletion.

[0296] In some embodiments, lymphodepletion is achieved by administering cyclophosphamide at about 50 mg / m 2 / day for 2 days, with fludarabine at approximately 20 mg / m 2 / day for 5 days, with cyclophosphamide and fludarabine both administered on the first 2 days, for a total of 5 days of lymphodepletion.

[0297] In some embodiments, lymphodepletion is achieved by administering cyclophosphamide at about 40 mg / m 2 / day for 2 days, with fludarabine at approximately 20 mg / m 2 / day for 5 days, with cyclophosphamide and fludarabine both administered on the first 2 days, for a total of 5 days of lymphodepletion.

[0298] In some embodiments, lymphodepletion is achieved by administering cyclophosphamide at about 40 mg / m 2 / day for 2 days, with fludarabine at approximately 15 mg / m 2 / day for 5 days, with cyclophosphamide and fludarabine both administered on the first 2 days, for a total of 5 days of lymphodepletion.

[0299] In some embodiments, lymphodepletion is achieved by administering cyclophosphamide at 60 mg / m 2 / day and fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 This is performed by administering the drug at a dose of 100 mg / day for 3 days.

[0300] In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, mesna is administered at 15 mg / kg. In some embodiments where mesna is infused, if infused continuously, mesna may be infused with cyclophosphamide for approximately 2 hours (on day -5 and / or day -4), then at a rate of 3 mg / kg / hour for the remaining 22 hours over a 24-hour period starting simultaneously with each cyclophosphamide dose.

[0301] In some embodiments, lymphodepletion comprises treating the patient with an IL-2 regimen beginning the day after administration of the third population of TILs to the patient.

[0302] In some embodiments, lymphodepletion comprises treating the patient with an IL-2 regimen beginning on the same day as administration of the third population of TILs to the patient.

[0303] In some embodiments, lymphodepletion comprises 5 days of preconditioning therapy. In some embodiments, the days are indicated as day -5 to day -1 or day 0 to day 4. In some embodiments, the regimen includes cyclophosphamide on day -5 and -4 (i.e., day 0 and day 1). In some embodiments, the regimen includes intravenous cyclophosphamide on day -5 and -4 (i.e., day 0 and day 1). In some embodiments, the regimen includes 60 mg / kg intravenous cyclophosphamide on day -5 and -4 (i.e., day 0 and day 1). In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the regimen further includes fludarabine. In some embodiments, the regimen further includes intravenous fludarabine. In some embodiments, the regimen includes 25 mg / m 2 In some embodiments, the regimen further comprises intravenous fludarabine at 25 mg / m on days -5 and -1 (i.e., days 0-4). 2 In some embodiments, the regimen further comprises intravenous fludarabine at 25 mg / m on days -5 and -1 (i.e., days 0-4). 2 of intravenous fludarabine.

[0304] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 5 days.

[0305] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 5 days.

[0306] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0307] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0308] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 1 day.

[0309] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0310] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0311] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 4.

[0312] [Table 5]

[0313] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 5.

[0314] [Table 6]

[0315] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 6.

[0316] [Table 7]

[0317] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 7.

[0318] [Table 8]

[0319] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 8.

[0320] [Table 9]

[0321] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 9.

[0322] [Table 10]

[0323] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 10

[0324] [Table 11]

[0325] In some embodiments, the non-myeloablative lymphodepletion regimen is administered according to Table 11.

[0326] [Table 12]

[0327] In some embodiments, the TIL infusion used in the foregoing embodiments of the myeloablative lymphodepletion regimen can be any TIL composition described herein, and can be administration of additional and combination therapies (such as PD-1 and / or PD-L1 inhibitors) with IL-2 regimens as described herein.

[0328] 2.IL-2 regimen In some embodiments, the IL-2 regimen includes a high-dose IL-2 regimen, which includes aldesleukin or a biosimilar or variant thereof, administered intravenously starting the day after administration of the therapeutically effective dose of the therapeutic TIL population, wherein aldesleukin or a biosimilar or variant thereof is administered using a 15-minute bolus intravenous infusion at a dose of 0.037 mg / kg or 0.044 mg / kg IU / kg (patient body weight) every 8 hours until tolerated, for a maximum of 14 doses. After a 9-day rest period, 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.

[0329] In some embodiments, the IL-2 regimen comprises a decrescendo IL-2 regimen, such as that 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 decrescendo IL-2 regimen comprises 18 x 10 IL-2 administered intravenously over 6 hours. 6 IU / m 2 Aldesleukin, or its biosimilar or variant, followed by 18 × 10 administered intravenously over 12 hours 6 IU / m 2 , followed by 18 × 10 intravenous injections over 24 hours 6 IU / m 2 , followed by 4.5 × 10 intravenous doses over 72 hours 6 IU / m 2 This treatment cycle may be repeated every 28 days for up to four cycles. In some embodiments, the decrescendo IL-2 regimen includes 18,000,000 IU / m on day 1. 2 , 9,000,000 IU / m on the second day 2 , 4,500,000 IU / m on days 3 and 4 2 Includes.

[0330] In some embodiments, the IL-2 regimen comprises 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. Immunology 2000, 19, 665-673; Hartemann, et al., Lancet Diabetes Endocrinol. 2013, 1, 295-305; and Rosenzwaig, et al., Ann. Rheum. Dis. 2019, 78, 209-217 (the disclosures of which are incorporated herein by reference). In some embodiments, the low-dose IL-2 regimen comprises m 2 Winning 18 x 106 IU of aldesleukin, or its biosimilar or variant, given as a continuous infusion every 24 hours for 5 days, followed by 2 to 6 days without IL-2 therapy, followed optionally by an additional 5 days of intravenous aldesleukin or its biosimilar or variant, given as a continuous infusion every 24 hours. 2 Winning 18 x 10 6 IU as a continuous infusion, optionally followed by 3 weeks without IL-2 therapy, after which additional cycles may be administered.

[0331] In some embodiments, IL-2 is administered at a maximum dose of up to six doses. In some embodiments, a high-dose IL-2 regimen is adapted for pediatric use. In some embodiments, a dose of 600,000 international units (IU) / kg of aldesleukin is used every 8-12 hours for up to six doses. In some embodiments, a dose of 500,000 international units (IU) / kg of aldesleukin is used every 8-12 hours for up to six doses. In some embodiments, a dose of 400,000 international units (IU) / kg of aldesleukin is used every 8-12 hours for up to six doses. In some embodiments, a dose of 500,000 international units (IU) / kg of aldesleukin is used every 8-12 hours for up to six doses. In some embodiments, a dose of 300,000 international units (IU) / kg of aldesleukin is used every 8-12 hours for up to six doses. In some embodiments, a dose of 200,000 International Units (IU) / kg of aldesleukin is used every 8-12 hours, up to a maximum of 6 doses. In some embodiments, a dose of 100,000 International Units (IU) / kg of aldesleukin is used every 8-12 hours, up to a maximum of 6 doses.

[0332] In some embodiments, the IL-2 regimen comprises administering pegylated IL-2 at a dose of 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bempegaldesleukin, or a fragment, variant, or biosimilar thereof, at a dose of 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days.

[0333] In some embodiments, the IL-2 regimen comprises administering THOR-707, or a fragment, variant, or biosimilar thereof, at a dose of 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days.

[0334] In some embodiments, the IL-2 regimen includes administration of nembareukin alfa, or a fragment, variant, or biosimilar thereof, after administration of TILs. In certain embodiments, the patient is administered nembareukin at a dose of 0.10 mg / day to 50 mg / day every 1, 2, 4, 6, 7, 14, or 21 days.

[0335] In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than wild-type IL-2 molecules, such as, but not limited to, aldesleukin (Proleukin®) or equivalent molecules.

[0336] In some embodiments, the TIL infusion used with the foregoing embodiments of the myeloablative lymphodepletion regimen may be any TIL composition described herein, and may also include infusion of MILs and PBLs in lieu of TIL infusion, as well as administration of additional and combination therapies (such as PD-1 and / or PD-L1 inhibitors and / or CTLA-4 inhibitors) with the IL-2 regimens described herein. [Example]

[0337] Embodiments encompassed herein will now be described with reference to the following examples, which are provided for illustrative purposes only, and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather as encompassing any and all variations that become evident as a result of the teachings provided herein.

[0338] Example 1: Exemplary Manufacturing Process for Gene-Edited TILs An exemplary TIL production process is shown in Figures 1A-F. Briefly, on day 0, tumor tissue in hypothermia is isolated, the bioburden sample is preserved in transport medium, and tumor fragments are seeded into multiple (2, 3, or 4) G-Rex 100 MCS flasks at a density of ≤50 fragments / flask. Excess fragments are snap-frozen. In some embodiments, an activation step may be incorporated into the pre-REP step, providing a better costimulatory environment for TILs within the rosette / tumor ME. For example, on day 3, 60 μg of OKT3 or TransAct is added to each of multiple G-Rex 100 MCS flasks, and the cells are subjected to the first activation. On day 7 / 8, the volume is reduced, the sample is filtered, and the pooled sample is transferred to the EXP1000. The sample is removed for cell counting / viability analysis. The cells were washed and centrifuged at 400 g for 10 min at 20°C, split into TALEN and control samples at a ratio of ≥9:1, and the cells were resuspended in T buffer and diluted to 10 × 10 6Each cuvette is electroporated with TALEN mRNA (TALEN sample) or no RNA (control sample). The electroporated control and TALEN samples from each cuvette are seeded into a G-Rex 100M flask containing 100 mL of culture medium containing 6000 IU / mL IL-2 and incubated at 37°C for 1 hour. Feeder cells are irradiated, thawed, pooled, and incubated with IL-2. Samples are removed for cell count and viability analysis. Feeder cells and additional IL-2 and OKT3 are added to the incubated control and TALEN samples in G-Rex 100MCS flasks to generate REP cultures (1x G-Rex 100MCS for control REP cultures, ≤9x G-Rex 100MCS for TALEN REP cultures). 500 mL of culture medium, along with 3000 IU / mL of IL-2, is added to each flask of the REP culture on days 10 / 11. Alternatively, the cell culture medium can be completely replaced with fresh culture medium. This step eliminates the need for cell suspension transfer and does not require the use of the G-Rex 500MCS. The volume is reduced on day 16 and the samples are pooled. The samples are transferred through a blood filter and removed for cell count / viability analysis. The control sample is centrifuged to generate a control final formulation, which is cryopreserved under controlled rate freezing. The TALEN sample is processed through the LOVO system to generate a TALEN retentate final formulation, which is cryopreserved under controlled rate freezing. The formulated product sample is then subjected to quality control analysis.

[0339] This process has the following advantages: a) eliminates the suspension transfer step for activation in bags, b) allows for 1 / 10 scale control, c) eliminates in-process transfer to G-Rex10, d) eliminates the overnight incubation step at 30°C in favor of immediate reactivation (REP) in ambient temperature medium, and e) eliminates one processing day.

[0340] Example 2: Abbreviated proliferation assay of gene-edited TILs The current IL-2-independent proliferation assay for gene-edited TILs takes 28 days. A study was conducted to determine whether the current IL-2-independent proliferation assay could be shortened to approximately 14 days or less.

[0341] Experimental design Two TALEN-edited TIL products from tumor samples L4346 and M1214, expanded for 18 and 22 days, were seeded at 2e6 cells / well in G-Rex 24-well plates on day 0 and cultured in cell culture medium consisting 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). Cell numbers were counted on days 7, 10, and 14 after initiating cell culture.

[0342] result Figure 2 shows the proliferation of TILs in terms of fold expansion. TILs cultured in the presence of IL-2 continued to proliferate after seeding, while TILs cultured in the absence of IL-2 showed minimal increase in fold expansion at day 7, but decreased cell numbers (less than 1-fold expansion) by day 14 in both experiments.

[0343] Example 3: Click-iT™ EdU proliferation assay using Jurkat cells An initial study was performed to test the Click-iT™ EdU proliferation assay conditions on Jurkat cells.

[0344] Experimental design Jurkat cells were seeded at 1e4, 1e5, 2e5, and 1e6 cells / mL on day 0 and cultured in the absence of IL-2. 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, 8, 6, 4, and 2 hours) before performing Click-iT™ EdU proliferation assays on days 1, 5, and 7 after culture initiation.

[0345] After incubation with EdU, 80 μl of medium was removed from each well, and 50 μl of Click-iT® EdU fixative was added to each well. The plate was incubated for 5 minutes at room temperature. The fixative was removed, and the wells were washed with 200 μl of Click-iT® EdU wash buffer. Then, 50 μl of Click-iT® EdU reaction cocktail, containing deionized water, Click-iT® EdU master mix, Click-iT® EdU reaction additive, and HRP-azide, was added to each well and incubated for 30 minutes at room temperature. The reaction cocktail was removed, and the plate was tapped dry on a paper towel. Next, 200 μl of 1.5% BSA blocking solution was added to each well, and the plate was incubated for 5 minutes at room temperature, protected from light. The blocking solution was removed, and the plate was washed three times with 200 μl of Click-iT® EdU wash buffer. 100 μl of Amplex™ UltraRed reaction mixture, containing Amplex™ UltraRed reaction buffer, Amplex™ UltraRed reagent, and hydrogen peroxide solution, was added to each well, and the plate was incubated at room temperature, protected from light, for 15 minutes. The reaction was stopped by adding 10 μl / well of Amplex™ UltraRed stop solution. The plate was then read on a fluorescence microplate reader.

[0346] result Figures 3 and 4 show that proliferation signals were observed at 10-20 μM EdU across all cell concentrations, with 1e3 to 1e4 cells / well being the optimal plating conditions.

[0347] Example 4: Click-iT™ EdU proliferation assay of gene-edited TILs A study was conducted to test the Click-iT™ EdU proliferation assay conditions on gene-edited TILs.

[0348] Experimental design Two PD-1 TALEN-edited TIL lines (L4340 and EP11231) were tested using the Click-iT™ EdU proliferation assay. The corresponding mock-edited TIL line was used as a negative control, and Jurkat cells were used as a positive control.

[0349] TILs were seeded at 1e5 / well and 2e5 / well and Jurkat cells were seeded at 2e3 / well in triplicate in cell culture medium without IL-2 on day 0. Serial dilutions of Jurkat cells at 4e5 / well, 1e5 / well, 2.5e4 / well, 6.25e4 / well, and 1.56e4 / well were also tested.

[0350] Click-iT™ EdU proliferation assay 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 performing the Click-iT™ EdU proliferation assay.

[0351] result Figure 5 shows that no proliferation was observed on days 7, 10, and 14 for the two PD-1 TALEN-edited TIL lines and the mock gene-edited TIL line.

[0352] Example 5: Click-iT™ EdU proliferation assay of additional gene-edited TIL lots Further studies were conducted to test the Click-iT™ EdU proliferation assay conditions on five additional gene-edited TILs.

[0353] Experimental design Five PD-1 TALEN-edited TIL lots (L4374, K7091, L4353, L4340, and EP11231) were tested using the Click-iT™ EdU proliferation assay. Jurkat cells were used as a positive control.

[0354] On day 0, TILs were seeded at 1e5 / well and Jurkat cells were seeded at 2e3 / well in triplicate in cell culture medium without IL-2.

[0355] Click-iT™ EdU proliferation assays at days 7 and 10 after seeding. EdU was added at a concentration of 20 μM 24 hours before performing the Click-iT™ EdU proliferation assay.

[0356] result Figure 6 shows that no proliferation was observed on days 7 and 10 for the five PD-1 TALEN-edited TIL lots.

[0357] The examples set forth above are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains.

[0358] All heading and section designations are used solely for clarity and reference purposes and should not be construed as limiting in any way, for example, those skilled in the art will understand the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention as described herein.

[0359] All references cited in this specification are 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.

[0360] As will be apparent to those skilled in the art, many modifications and variations of this application may be made without departing from the spirit and scope thereof. The specific embodiments and examples described herein are offered by way of example only, and this application should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. 1. A method for assessing the proliferation index of a gene-edited T cell population, comprising: (a) culturing a first portion of the gene-edited T cell population in cell culture medium that does not contain IL-2; (b) adding an agent to the cell culture medium, wherein the agent is incorporated into the DNA of cells during proliferation; (c) measuring the amount of the agent incorporated into the gene-edited T cells at one or more time points; (d) calculating the proliferation index of the gene-edited T cell population according to the amount of the agent incorporated into the gene-edited T cells at the one or more time points.

2. The agent is 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 3. The method of claim 1, wherein the modified nucleotide is selected from the group consisting of:

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

4. 4. The method of claim 1, wherein measuring the amount of the agent incorporated into the T cells comprises performing a click reaction using HRP.

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

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

7. 7. The method of any one of claims 1 to 6, wherein the one or more time points are selected from the group consisting of: 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 day, and 28 day.

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

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

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

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

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

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

14. 14. The method of claim 13, wherein the transformed T cell is a Jurkat cell.

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

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

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

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

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

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

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

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

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

24. 17. The method of Claim 15 or 16, wherein the gene-edited TIL comprises a first TALE nuclease system for regulating expression of a first protein and a second TALE nuclease system for regulating expression of a second protein.

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

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

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

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

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

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

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

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

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

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

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

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

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

38. The CAR of the CAR T cells is 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) antibody Gen; 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); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropein) subunit, beta, 9 (LMP2); glycoprotein 100 (gp100); breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Ab l), an oncogene fusion protein (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 class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary 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 K9 (LY6K); olfactory receptor 51 E2 (OR51 E2); TCR gamma alternative reading frame protein (TARP); Wilms' tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAG E-1 a); melanoma-associated antigen 1 (MAG E-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm 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; surviving; telomerase; prostate cancer tumor antigen-1, melanoma antigen 1 recognized by T cells; rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA1 7); 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 1 B1 (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); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mutated) 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).

39. 1. A method for treating a cancer patient, said method comprising: (a) generating a gene-edited TIL population from a tumor resected from said cancer patient; (b) assessing the proliferation index of the gene-edited TIL population using the method of any one of claims 1 to 36; and (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 positive control.

40. 40. The method of 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)), renal cancer, and renal cell carcinoma.

41. The therapeutically effective dose of the gene-edited TILs is about 1×10 ９ ~Approx. 1×10 １１ 41. The method of claim 39 or 40, wherein the TIL is a TIL.

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

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

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

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

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

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

48. 48. The method of any one of claims 39-47, wherein the gene editing silences or reduces expression of one or more immune checkpoint genes in at least a portion of the gene-edited TIL population.

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

Images