A two-dimensional process for the expansion of tumor-infiltrating lymphocytes and subsequent therapeutic approaches

Abstract

Provided herein is the method for producing TIL through (i) pre-REP amplification step, (ii) co-culture with dendritic cells that present tumor-specific antigen, (iii) co-culture with tumor-derived organoid, (iv) selection of tumor-reactive TIL, and (v) rapid expansion of the tumor-reactive TIL, or combination thereof.The therapeutic and diagnostic materials that result are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 384,576, filed November 21, 2022, the entire disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Treatment of large, refractory cancers using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. TILs are predominantly T cells, and IL-2-based TIL expansion, followed by the "rapid expansion process" (REP), has become the preferred method for TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. Numerous approaches to improve response to TIL therapy in melanoma and to extend TIL therapy to other tumor types have been investigated with limited success, and the field remains challenging. Goff, et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al., Clin. Cancer Res. 2011, 17, 4550-57. Combination studies with single immune checkpoint inhibitors have also been described, but further research is ongoing and additional treatment options are needed (Kverneland, et al., Oncotarget, 2020, 11(22), 2092-2105).

[0003] Although TILs naturally infiltrate tumors, reports suggest that a large number of these are "bystander TILs" that do not respond to tumor-specific neoantigens. Gokuldass, A., et al. Cancers, 2020, 12(11), 3344. This unbalanced and untargeted expansion regimen can result in a disproportionate amount of bystander TILs in the therapeutic population. There remains a need in the art for methods to identify tumor-reactive TILs within a population of TILs and specifically expand this tumor-reactive subpopulation. The present disclosure fulfills this need by providing methods for identifying and expanding tumor-reactive TIL subpopulations and compositions derived therefrom. Summary of the Invention

[0004] Provided herein are methods for enriching and identifying tumor-reactive cells in a tumor sample so that the tumor-reactive cells can be distinguished from bystander TILs. In one aspect, the disclosure herein further includes a method for expanding previously identified tumor-reactive TILs.

[0005] In some embodiments, the method comprises: (a) obtaining a tumor sample from a patient; (b) dividing the tumor sample into a first portion and a second portion; (c) performing a first expansion of the first population of TILs in the second portion of the tumor sample by culturing the second portion of the tumor sample in the first cell culture medium and IL-2 to produce a second population of TILs; and (d) contacting the second population of TILs with tumor cells or tumor cell antigens from or derived from a tumor digest obtained by digestion of the first portion of the tumor sample to produce a third population of TILs. Including, The third population of TILs comprises a plurality of tumor-reactive TILs that are enriched compared to the second population of TILs.

[0006] In some embodiments, step (d) is carried out for about 1 to about 3 days.

[0007] In some embodiments, the method comprises: (a) obtaining a tumor sample from a patient; (b) dividing the tumor sample into a first portion and a second portion; (c) performing a first expansion of the first population of TILs in the second portion of the tumor sample by culturing the second portion of the tumor sample in the first cell culture medium and IL-2 to produce a second population of TILs; and (d) contacting the second population of TILs with tumor cells or tumor cell antigens from a tumor digest obtained by digestion of a first portion of the tumor sample or with a population of mature dendritic cells (DCs) generated from culturing a population of immature DCs with tumor cells or tumor cell antigens derived from the tumor digest to generate a third population of TILs, wherein the third population of TILs comprises a plurality of tumor-reactive TILs enriched compared to the second population of TILs.

[0008] In some embodiments, the population of immature DCs is generated by culturing a population of monocytes in the presence of GM-CSF and IL-4. In some embodiments, the population of monocytes is obtained from PBMCs. In some embodiments, the PBMCs are obtained from a patient. In some embodiments, the population of monocytes is cultured in the presence of GM-CSF and IL-4 for about 6 days. In some embodiments, culturing the population of immature DCs in the presence of tumor cells or tumor cell antigens from or derived from a tumor digest comprises generating a tumor lysate from the tumor digest and culturing immature DCs in the presence of tumor cells or tumor cell antigens from or derived from the tumor lysate. In some embodiments, the population of immature DCs is cultured with tumor cells from the tumor digest at a ratio of about 3:1. In some embodiments, the population of immature DCs is cultured in the presence of tumor cells or tumor cell antigens from or derived from a tumor digest for about 24 hours. In some embodiments, the tumor digest or tumor lysate is subjected to dead cell removal before culturing with the population of immature DCs. In some embodiments, culturing the population of immature DCs in the presence of tumor cells or tumor cell antigens from or derived from the tumor digest is performed in the presence of TNFα, IL-6, and IL-1β. In some embodiments, the concentration of TNFα is about 2000 IU / ml. In some embodiments, the concentration of IL-6 is about 2000 IU / ml. In some embodiments, the concentration of IL-1β is about 400 IU / ml. In some embodiments, a second population of TILs is cultured with mature DCs. In some embodiments, step (d) is performed for about 1 to 3 days.

[0009] In some embodiments, the method comprises: (a) obtaining a tumor sample from a patient; (b) dividing the tumor sample into a first portion and a second portion; (c) performing a first expansion of the first population of TILs in the second portion of the tumor sample by culturing the second portion of the tumor sample in the first cell culture medium and IL-2 to produce a second population of TILs; and (d) contacting the second population of TILs with organoids generated from the first portion of the tumor sample to generate a third population of TILs, wherein the third population of TILs comprises a plurality of tumor-reactive TILs enriched compared to the second population of TILs.

[0010] In some embodiments, generating organoids from the first portion of the tumor sample includes digesting the first portion of the tumor sample to obtain a tumor digest, and generating organoids from the tumor digest.

[0011] In some embodiments, generating organoids comprises: (a) propelling a first portion of the tumor sample and an unpolymerized liquid matrix material through one or more channels of a microfluidic device; i. the microfluidic device controls the pressure, flow rate, or pressure and flow rate in one or more channels and maintains a temperature of 20°C or less, such that tumor-derived cells or a plurality of tumor fragments and an unpolymerized liquid matrix in the tumor sample move through the one or more channels in a laminar flow; (b) combining the tumor-derived cells or a plurality of tumor fragments and an unpolymerized liquid matrix material in a microfluidic device to form a plurality of droplets of the unpolymerized mixture; and (c) exposing the plurality of droplets of the unpolymerized mixture to a temperature greater than 25°C to polymerize the liquid matrix material and form organoids; Includes.

[0012] In some embodiments, the methods disclosed herein further include identifying a plurality of tumor-reactive TILs in a third population of TILs. In some embodiments, identifying a plurality of tumor-reactive TILs includes determining whether the TILs exhibit an activation signal that identifies the TILs as tumor-reactive. In some embodiments, the activation signal includes increased and / or decreased cell surface expression of one or more proteins. In some embodiments, the one or more proteins are selected from the group consisting of CD3, CD4, CD8, PD-1, LAG3, Tim3, TIGIT, CD103, CD39, CD134, CD137, CD25, CD69, HLA-DR, CD107a, CD40L, Ki46, CD45RA, CCR7, and KLRG1. In some embodiments, the cell surface expression of the one or more proteins is determined by flow cytometry. In some embodiments, flow cytometry is performed using a SONY FX 500, Miltenyi Tyto, or Miltenyi CliniMACS flow-activated cell sorter. In some embodiments, the activation signal comprises cell morphology. In some embodiments, the cell morphology is a flattened, rounded cell morphology. In some embodiments, the activation signal is the concentration of mitochondrial mass adjacent to the plasma membrane of TILs. In some embodiments, the activation signal is determined by an imaging-based cell separation method.

[0013] In some embodiments, the methods disclosed herein further include collecting the identified plurality of tumor-reactive TILs. In some embodiments, collecting the plurality of tumor-reactive TILs includes separating the plurality of tumor-reactive TILs from non-tumor-reactive TILs in a third population of TILs. In some embodiments, separating the plurality of tumor-reactive TILs includes removing non-tumor-reactive TILs from the third population of TILs.

[0014] In some embodiments, the methods disclosed herein include: (e) performing a second expansion by culturing the third population of TILs or the collected plurality of tumor-reactive TILs in a second cell culture medium supplemented with additional IL-2, OKT-3, and antigen-presenting cells to generate a fourth population of TILs. The method further includes performing the following steps.

[0015] In some embodiments, the first portion of the tumor sample comprises approximately one-third of the tumor sample. In some embodiments, the second portion of the tumor sample comprises approximately one-half of the tumor sample. In some embodiments, the second portion of the tumor sample comprises approximately one-third of the tumor sample. In some embodiments, the tumor digest is subjected to 1, 2, 3, 4, 5, or 10 freeze-thaw cycles. In some embodiments, the first portion of the tumor sample comprises at least 3 million cells. In some embodiments, steps (a)-(e) are performed within a period of about 17 to about 24 days, within a period of about 18 to about 22 days, within a period of about 20 to about 22 days, or within a period of about 22 days. In some embodiments, the first cell culture medium further comprises a factor selected from the group consisting of IL-7, IL-15, IL-21, IL-12, leukemia inhibitory factor (LIF), beta fibroblast growth factor (bFGF), and combinations thereof. In some embodiments, the second cell culture medium further comprises a factor selected from the group consisting of IL-7, IL-15, IL-21, IL-12, LIF, bFGF, 41BBL, OX40L, CD86, CD64, and combinations thereof. In some embodiments, the tumor sample is selected from the group consisting of a solid tumor, a fine needle aspirate, and a mini-biopsy. In some embodiments, the activating signal comprises an increase in secreted interferon gamma (IFNγ). In some embodiments, the cell culture medium is provided in a container selected from the group consisting of a G-container and a Xuri cellbag.

[0016] In some embodiments, the methods disclosed herein further comprise gene editing the second population of TILs, the third population of TILs, or the plurality of tumor-reactive TILs.

[0017] Further provided herein is a pharmaceutical composition for the treatment of cancer, comprising a fourth population of TILs generated using the methods disclosed herein.

[0018] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma. In some embodiments, the pharmaceutical composition further comprises a cryopreservation material. In some embodiments, the cryopreservation material comprises dimethyl sulfoxide. In some embodiments, the pharmaceutical composition further comprises a cryopreservation material and an isotonicity agent. In some embodiments, the pharmaceutical composition further comprises a cryopreservation material comprising dimethyl sulfoxide and an isotonicity agent comprising sodium chloride, sodium gluconate, and sodium acetate. In some embodiments, the pharmaceutical composition further comprises a cryopreservation material comprising dimethyl sulfoxide and dextran 40 and an isotonicity agent comprising sodium chloride, sodium gluconate, and sodium acetate. In some embodiments, the composition is provided in a sterile infusion bag. In some embodiments, the composition is harvested using the LOVO Cell Processing System. In some embodiments, the TILs are gene edited. [Brief explanation of the drawings]

[0019] [Figure 1] 1 illustrates an embodiment of a process for enriching tumor-reactive TILs using autologous tumor digest. [Figure 2] Illustrated are embodiments of a process for enriching tumor-reactive TILs using autologous dendritic cells (DCs) or DC-like cells. [Figure 3] 1 illustrates an embodiment of a process for enriching tumor-reactive TILs using autologous organoids / tumor organoids. [Figure 4A] Results from TIL:tumor cell co-cultures are shown. [Figure 4B] Results from TIL:tumor cell co-cultures are shown.

[0020] Brief description of sequence listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0021] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0022] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0023] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0024] SEQ ID NO: 5 is an IL-2 form.

[0025] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0026] SEQ ID NO: 7 is an IL-2 form.

[0027] SEQ ID NO: 8 is a mucin domain polypeptide.

[0028] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0029] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0030] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0031] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0032] SEQ ID NO: 13 is the IL-2 sequence.

[0033] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0034] SEQ ID NO: 15 is the IL-2 mutein sequence.

[0035] SEQ ID NO: 16 is HCDR1_IL-2 for IgG.IL2R67A.H1.

[0036] SEQ ID NO: 17 is HCDR2 for IgG.IL2R67A.H1.

[0037] SEQ ID NO: 18 is the HCDR3 for IgG.IL2R67A.H1.

[0038] SEQ ID NO: 19 is the HCDR1_IL-2 kabat for IgG.IL2R67A.H1.

[0039] SEQ ID NO: 20 is the HCDR2 kabat for IgG.IL2R67A.H1.

[0040] SEQ ID NO: 21 is the HCDR3 kabat for IgG.IL2R67A.H1.

[0041] SEQ ID NO: 22 is the HCDR1_IL-2 sequence for IgG.IL2R67A.H1.

[0042] SEQ ID NO: 23 is the HCDR2 region for IgG.IL2R67A.H1.

[0043] SEQ ID NO: 24 is the HCDR3 region for IgG.IL2R67A.H1.

[0044] SEQ ID NO: 25 is HCDR1_IL-2 IMGT for IgG.IL2R67A.H1.

[0045] SEQ ID NO: 26 is the HCDR2 IMGT for IgG.IL2R67A.H1.

[0046] SEQ ID NO: 27 is the HCDR3 IMGT for IgG.IL2R67A.H1.

[0047] SEQ ID NO: 28 is the VH chain for IgG.IL2R67A.H1.

[0048] SEQ ID NO: 29 is the heavy chain for IgG.IL2R67A.H1.

[0049] SEQ ID NO: 30 is the LCDR1 kabat for IgG.IL2R67A.H1.

[0050] SEQ ID NO: 31 is the LCDR2 kabat for IgG.IL2R67A.H1.

[0051] SEQ ID NO: 32 is the LCDR3 kabat for IgG.IL2R67A.H1.

[0052] SEQ ID NO: 33 is the LCDR1 chothia for IgG.IL2R67A.H1.

[0053] SEQ ID NO: 34 is the LCDR2 sequence for IgG.IL2R67A.H1.

[0054] SEQ ID NO: 35 is the LCDR3 chothia for IgG.IL2R67A.H1.

[0055] SEQ ID NO: 36 is the VL chain.

[0056] SEQ ID NO: 37 is the light chain.

[0057] SEQ ID NO: 38 is the light chain.

[0058] SEQ ID NO: 39 is the light chain.

[0059] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0060] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0061] SEQ ID NO: 42 is the heavy chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0062] SEQ ID NO: 43 is the light chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0063] SEQ ID NO: 44 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0064] SEQ ID NO: 45 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0065] SEQ ID NO: 46 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0066] SEQ ID NO: 47 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0067] SEQ ID NO: 48 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0068] SEQ ID NO: 49 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0069] SEQ ID NO: 50 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0070] SEQ ID NO: 51 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0071] SEQ ID NO: 52 is the heavy chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0072] SEQ ID NO: 53 is the light chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0073] SEQ ID NO: 54 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0074] SEQ ID NO: 55 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0075] SEQ ID NO: 56 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0076] SEQ ID NO: 57 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0077] SEQ ID NO: 58 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0078] SEQ ID NO: 59 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0079] SEQ ID NO: 60 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0080] SEQ ID NO: 61 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0081] SEQ ID NO: 62 is the Fc domain for the TNFRSF agonist fusion protein.

[0082] SEQ ID NO: 63 is the linker for the TNFRSF agonist fusion protein.

[0083] SEQ ID NO: 64 is the linker for the TNFRSF agonist fusion protein.

[0084] SEQ ID NO: 65 is the linker for the TNFRSF agonist fusion protein.

[0085] SEQ ID NO: 66 is the linker for the TNFRSF agonist fusion protein.

[0086] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0087] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0088] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0089] SEQ ID NO: 70 is the linker for the TNFRSF agonist fusion protein.

[0090] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0091] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0092] SEQ ID NO: 73 is the Fc domain for the TNFRSF agonist fusion protein.

[0093] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0094] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0095] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0096] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0097] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0098] SEQ ID NO: 79 is the heavy chain variable region (VH) for 4-1BB agonist antibody 4B4-1-1 version 1.

[0099] SEQ ID NO: 80 is the light chain variable region (VL) for 4-1BB agonist antibody 4B4-1-1 version 1.

[0100] SEQ ID NO: 81 is the heavy chain variable region (VH) for 4-1BB agonist antibody 4B4-1-1 version 2.

[0101] SEQ ID NO: 82 is the light chain variable region (VL) for 4-1BB agonist antibody 4B4-1-1 version 2.

[0102] SEQ ID NO: 83 is the heavy chain variable region (VH) for the 4-1BB agonist antibody H39E3-2.

[0103] SEQ ID NO: 84 is the light chain variable region (VL) for the 4-1BB agonist antibody H39E3-2.

[0104] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0105] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0106] SEQ ID NO: 87 is the heavy chain for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0107] SEQ ID NO: 88 is the light chain for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0108] SEQ ID NO: 89 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0109] SEQ ID NO: 90 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0110] SEQ ID NO: 91 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0111] SEQ ID NO: 92 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0112] SEQ ID NO: 93 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0113] SEQ ID NO: 94 is the light chain CDR1 for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0114] SEQ ID NO: 95 is the light chain CDR2 for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0115] SEQ ID NO: 96 is the light chain CDR3 for the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0116] SEQ ID NO: 97 is the heavy chain for the OX40 agonist monoclonal antibody 11D4.

[0117] SEQ ID NO: 98 is the light chain for the OX40 agonist monoclonal antibody 11D4.

[0118] SEQ ID NO: 99 is the heavy chain variable region (VH) for OX40 agonist monoclonal antibody 11D4.

[0119] SEQ ID NO: 100 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 11D4.

[0120] SEQ ID NO: 101 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody 11D4.

[0121] SEQ ID NO: 102 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 11D4.

[0122] SEQ ID NO: 103 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 11D4.

[0123] SEQ ID NO: 104 is the light chain CDR1 for OX40 agonist monoclonal antibody 11D4.

[0124] SEQ ID NO: 105 is the light chain CDR2 for OX40 agonist monoclonal antibody 11D4.

[0125] SEQ ID NO: 106 is the light chain CDR3 for OX40 agonist monoclonal antibody 11D4.

[0126] SEQ ID NO: 107 is the heavy chain for the OX40 agonist monoclonal antibody 18D8.

[0127] SEQ ID NO: 108 is the light chain for the OX40 agonist monoclonal antibody 18D8.

[0128] SEQ ID NO: 109 is the heavy chain variable region (VH) for OX40 agonist monoclonal antibody 18D8.

[0129] SEQ ID NO: 110 is the light chain variable region (VL) for OX40 agonist monoclonal antibody 18D8.

[0130] SEQ ID NO: 111 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody 18D8.

[0131] SEQ ID NO: 112 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 18D8.

[0132] SEQ ID NO: 113 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 18D8.

[0133] SEQ ID NO: 114 is the light chain CDR1 for OX40 agonist monoclonal antibody 18D8.

[0134] SEQ ID NO: 115 is the light chain CDR2 for the OX40 agonist monoclonal antibody 18D8.

[0135] SEQ ID NO: 116 is the light chain CDR3 for OX40 agonist monoclonal antibody 18D8.

[0136] SEQ ID NO: 117 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu119-122.

[0137] SEQ ID NO: 118 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu119-122.

[0138] SEQ ID NO: 119 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody Hu119-122.

[0139] SEQ ID NO: 120 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu119-122.

[0140] SEQ ID NO: 121 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu119-122.

[0141] SEQ ID NO: 122 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu119-122.

[0142] SEQ ID NO: 123 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu119-122.

[0143] SEQ ID NO: 124 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu119-122.

[0144] SEQ ID NO: 125 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu106-222.

[0145] SEQ ID NO: 126 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu106-222.

[0146] SEQ ID NO: 127 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody Hu106-222.

[0147] SEQ ID NO: 128 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu106-222.

[0148] SEQ ID NO: 129 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu106-222.

[0149] SEQ ID NO: 130 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu106-222.

[0150] SEQ ID NO: 131 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu106-222.

[0151] SEQ ID NO: 132 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu106-222.

[0152] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0153] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0154] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0155] SEQ ID NO: 136 is the heavy chain variable region (VH) for OX40 agonist monoclonal antibody 008.

[0156] SEQ ID NO: 137 is the light chain variable region (VL) for OX40 agonist monoclonal antibody 008.

[0157] SEQ ID NO: 138 is the heavy chain variable region (VH) for OX40 agonist monoclonal antibody 011.

[0158] SEQ ID NO: 139 is the light chain variable region (VL) for OX40 agonist monoclonal antibody 011.

[0159] SEQ ID NO: 140 is the heavy chain variable region (VH) for OX40 agonist monoclonal antibody 021.

[0160] SEQ ID NO: 141 is the light chain variable region (VL) for OX40 agonist monoclonal antibody 021.

[0161] SEQ ID NO: 142 is the heavy chain variable region (VH) for OX40 agonist monoclonal antibody 023.

[0162] SEQ ID NO: 143 is the light chain variable region (VL) for OX40 agonist monoclonal antibody 023.

[0163] SEQ ID NO: 144 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[0164] SEQ ID NO: 145 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[0165] SEQ ID NO: 146 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[0166] SEQ ID NO: 147 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[0167] SEQ ID NO: 148 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0168] SEQ ID NO: 149 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0169] SEQ ID NO: 150 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0170] SEQ ID NO: 151 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0171] SEQ ID NO: 152 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0172] SEQ ID NO: 153 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[0173] SEQ ID NO: 154 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0174] SEQ ID NO: 155 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[0175] SEQ ID NO: 156 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[0176] SEQ ID NO: 157 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[0177] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0178] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0179] SEQ ID NO: 160 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor nivolumab.

[0180] SEQ ID NO: 161 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor nivolumab.

[0181] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0182] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0183] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0184] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0185] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0186] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0187] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0188] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0189] SEQ ID NO: 170 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0190] SEQ ID NO: 171 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0191] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0192] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0193] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0194] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0195] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0196] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0197] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0198] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0199] SEQ ID NO: 180 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor durvalumab.

[0200] SEQ ID NO: 181 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor durvalumab.

[0201] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0202] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0203] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0204] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0205] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0206] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0207] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0208] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0209] SEQ ID NO: 190 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor avelumab.

[0210] SEQ ID NO: 191 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor avelumab.

[0211] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0212] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0213] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0214] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0215] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0216] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0217] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0218] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0219] SEQ ID NO: 200 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0220] SEQ ID NO: 201 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0221] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0222] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0223] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0224] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0225] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0226] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0227] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0228] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0229] SEQ ID NO: 210 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0230] SEQ ID NO: 211 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0231] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0232] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0233] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0234] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0235] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0236] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0237] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0238] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0239] SEQ ID NO: 220 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0240] SEQ ID NO: 221 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0241] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0242] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0243] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0244] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0245] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0246] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0247] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0248] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0249] SEQ ID NO: 230 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0250] SEQ ID NO: 231 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0251] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0252] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0253] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0254] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0255] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0256] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0257] 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.

[0258] The terms "co-administration," "co-administering," "administered in conjunction with," "administering in conjunction with," "simultaneous," and "concurrent," as used herein, encompass the administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the present invention) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0259] The term "in vivo" refers to events that take place within the body of a subject.

[0260] The term "in vitro" refers to events that take place outside the body of a subject. In vitro assays encompass cell-based assays in which live or dead cells are used, and can also encompass cell-free assays in which no intact cells are used.

[0261] The term "ex vivo" refers to events involving the performance of a treatment or procedure on 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 procedure.

[0262] The term "rapid amplification" 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 one-week period, 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 one-week period, or most preferably about 100-fold over a period of at least one week. A number of rapid amplification protocols are described herein.

[0263] By "tumor infiltrating lymphocytes" or "TILs" herein is meant a population of cells originally 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 primary TILs 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.

[0264] By "cell population" (including TILs) herein is meant a large number of cells sharing a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial proliferation of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP amplification typically yields a population of bulk TILs of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0265] By "cryopreserved TILs" herein is meant either primary TILs, bulk TILs, or expanded TILs (REP TILs) that have been processed and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs.

[0266] 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 a higher temperature (including, but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient).

[0267] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve 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 a patient.

[0268] The term "cryopreservation medium(s)" refers to any medium that can be used for the cryopreservation of 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.

[0269] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高 Central memory T cells refer to a subset of T cells that constitutively express CD4+, CD3+, CD127 (IL-7R), and IL-15R. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0270] The term "effector memory T cells" refers to T cells that, like central memory T cells, are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低), refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines (including interferon-γ, IL-4, and IL-5) following antigen stimulation. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lungs, liver, and gastrointestinal tract in humans. CD8+ effector memory T cells possess high amounts of perforin.

[0271] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Examples of closed systems include, but are not limited to, closed G-containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0272] The terms "fragment," "fragment," and "fragmented," as used herein to describe a process for destroying tumors, include mechanical fragmentation methods (such as crushing, slicing, dividing, and mincing tumor tissue), as well as any other method that disrupts the physical structure of tumor tissue.

[0273] 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.

[0274] 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 for a T cell phenotype (e.g., a CD3+ CD45+ T cell phenotype).

[0275] The term "anti-CD3 antibody" refers to an antibody or variant thereof (e.g., a monoclonal antibody), including a human antibody, a humanized antibody, a chimeric antibody, or a murine antibody, directed against the CD3 receptor in the T cell antigen receptor of a mature T cell. 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.

[0276] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof, including a human, humanized, chimeric, or murine antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells, 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 variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited at the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0277] [Table 1]

[0278] 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, their 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 provided in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), and forms of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b), as well as other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 refers to the PEGylated IL-2 prodrug bempegaldesleukin (NKTR-214, a PEGylated human recombinant IL-2 of SEQ ID NO: 4, in which an average of six lysine residues are N-substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl, as described herein. 6The present invention also encompasses PEGylated forms of IL-2, including PEG-substituted IL-2, bempegaldesleukin, which is available from Nektar Therapeutics, South San Francisco, CA, USA, or can be prepared by methods known in the art, such as the method described in Example 19 of WO 2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. 2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Bempegaldesleukin (NKTR-214) and other PEGylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. 2014 / 0328791 A1 and WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0279] In some embodiments, a suitable form of IL-2 for use in the present invention is THOR-707 (available from Synthorx, Inc.) The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 2020 / 0181220A1 and 2020 / 0330601A1, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is an interleukin-2 (IL-2) conjugate comprising an isolated and purified IL-2 polypeptide; and a conjugate moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, where the numbering of the amino acid residues corresponds to SEQ ID NO: 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine.In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid is N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isoprenoids, phenylalanine ... and selenocysteine, or selenocysteine. In some embodiments, the IL-2 conjugate has reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to the wild-type IL-2 polypeptide, hi some embodiments, the reduction in affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than a 99% reduction in binding affinity to IL-2Rα compared to the wild-type IL-2 polypeptide.In some embodiments, the decrease in affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or more, compared to the wild-type IL-2 polypeptide. In some embodiments, the conjugate moiety impairs or blocks binding of IL-2 to IL-2Rα. In some embodiments, the conjugate moiety comprises a water-soluble polymer. In some embodiments, the additional conjugate moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises a polyamine. In some embodiments, the conjugate moiety comprises a protein. In some embodiments, the additional conjugate moiety comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion.In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugate moiety comprises a polypeptide. In some embodiments, the additional conjugate moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugate moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugate moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker.In some embodiments, the homo-homobifunctional linker is selected from the group consisting of Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′-dithiobispropionimidate (DTBP), 1,4-di-(3′-(2′-pyridyl dithiobispropionimidate ... Thio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB) (e.g., 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BAS ED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker.In some embodiments, the heterobifunctional linker is N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide] xanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl (4-iodoacteyl)aminobenzoate ester (sIAB), sulfosuccinimidyl (4-iodoactyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide esters (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (s IAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive crosslinkers and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleic acid). Imidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azido) N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (A NB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl- 1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP),The linker may comprise benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleimide group, optionally including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative or analog thereof. In some embodiments, the conjugate moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugate moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in the present invention is PEGylated as disclosed in U.S. Patent Application Publication Nos. 2020 / 0181220A1 and 2020 / 0330601A1. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugate moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide iscomprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5; AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with reference to the amino acid position in SEQ ID NO: 5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue compared to SEQ ID NO: 5. In some embodiments, IL-2 forms suitable for use in the present invention lack engagement of the IL-2Rα chain but retain normal binding to the IL-2Rβγ signaling complex with intermediate affinity. In some embodiments, an IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugate moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; and AzK replaces an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, with reference to the amino acid position within SEQ ID NO:5. In some embodiments, an IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugate moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5; and AzK replaces an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72, with reference to the amino acid position within SEQ ID NO:5. In some embodiments, an IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugate moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5; AzK is located at positions K35, F42, F44, K43, E62, P65,Substitute amino acids at R38, T41, E68, Y45, V69, or L72.

[0280] In some embodiments, a suitable form of IL-2 for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a human interleukin-2 fragment (1-59), variant (Cys 125 >Ser 51 ) is a peptidyl linker ( 60 GG 61 ) to the human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS 138 ), produced in Chinese hamster ovary (CHO) cells, and also known as glycosylated human interleukin 2 (IL-2) (75-133)-peptide [Cys 125Nemvaleukin alpha is also known as the (51)>Ser]-mutant (1-59) fused to human interleukin 2 (IL-2) (4-74)-peptide (62-132) via a G2 peptide linker (60-61) and to human interleukin 2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) via a GSG3S peptide linker (133-138), and produced in Chinese hamster ovary (CHO) cells in glycoform alpha. The amino acid sequence of nemvaleukin alpha is given in SEQ ID NO: 6. In some embodiments, nembareukin alfa exhibits the following post-translational modifications: disulfide bridges at positions 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbering in SEQ ID NO: 6), and glycosylation sites at positions N187, N206, T212 (using the numbering in SEQ ID NO: 6). The preparation and properties of nembareukin alfa, and additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. 2021 / 0038684A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence given in SEQ ID NO: 6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO: 7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO: 7, or a variant, fragment, or derivative thereof.Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity of ILRα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, wherein the half-life of the fusion protein is improved compared to fusing the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker.

[0281] [Table 2-1] [Table 2-2]

[0282] In some embodiments, forms of IL-2 suitable for use in the present invention include a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ); the light chain variable region (V L ); and V H or V L In some embodiments, the antibody cytokine grafted protein comprises an IL-2 molecule or a fragment thereof grafted into the CDRs of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H); the light chain variable region (V L ); and V H or V L and a CDR-grafted antibody cytokine graft protein comprising an IL-2 molecule or fragment thereof grafted into the CDRs of: H or V L or a fragment thereof grafted into the CDR of SEQ ID NO: 37; an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38; an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29; an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 29; and an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

[0283] In some embodiments, the IL-2 molecule or fragment thereof is V H In some embodiments, the IL-2 molecule or a fragment thereof is grafted into HCDR1 of V H In some embodiments, the IL-2 molecule or a fragment thereof is grafted into the HCDR2 of V H In some embodiments, the IL-2 molecule or a fragment thereof is grafted into the HCDR3 of V LIn some embodiments, the IL-2 molecule or a fragment thereof is grafted into LCDR1 of V L In some embodiments, the IL-2 molecule or a fragment thereof is grafted into the LCDR2 of V L The IL-2 molecule is a mutant protein grafted into the LCDR3 of

[0284] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0285] In some embodiments, the IL-2 molecule is directly grafted into the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted into the CDR with a peptide linker and one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0286] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some instances, the IL-2 mutein includes an R67A substitution. In some embodiments, the IL-2 mutein includes the amino acid sequence SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein includes the amino acid sequence in Table 1 in U.S. Patent Application Publication No. 2020 / 0270334A1 (the disclosure of which is incorporated herein by reference).

[0287] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H In some embodiments, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 36. L In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 28. H V comprising the region and the amino acid sequence of SEQ ID NO: 36 LIn some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334A1, or a variant, derivative, or fragment thereof, or a conservative amino acid substitution thereof, or a protein with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant proteins described herein comprises the immunoglobulin sequence, framework sequence, or CDR sequence of palivizumab. 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 or comparable molecules). In some embodiments, the antibody cytokine transplant proteins described herein have the sequences shown in Table 3.

[0288] [Table 3-1] [Table 3-2] [Table 3-3]

[0289] 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 of B cells to express IgE and IgG1. 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 given in Table 2 (SEQ ID NO: 9).

[0290] 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 and from 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 an IL-7 receptor α-chain receptor and a common γ-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 recombinant human IL-7 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 10).

[0291] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15, which includes all forms of IL-2, including human and mammalian forms, their conservative amino acid substitutions, glycoforms, biosimilars, and variants. 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, unglycosylated 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 a recombinant human IL-15 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 11).

[0292] The term "IL-21" (also referred to herein as "IL21") refers to the multifunctional cytokine protein known as interleukin-21, which includes all forms of IL-21, including human and mammalian forms, their conservative amino acid substitutions, glycoforms, biosimilars, and variants. 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, unglycosylated 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 given in Table 2 (SEQ ID NO: 21).

[0293] The term "IL-15R agonist" (also referred to herein as "IL-15 agonist") refers to a molecule that activates the IL-15 signaling pathway through binding to the IL-15 receptor (IL-15R) β subunit and common γ (γC) subunit. IL-15 functions via a transpresentation mechanism in which IL-15 is presented on the surface of dendritic cells or other cells in a complex with the membrane-bound α-subunit of the IL-15 receptor (IL-15Rα), and this complex interacts with the IL-15R β and γC subunits expressed on NK cells, NKT cells, or T cells. See Stonier and Schluns, Immunol Lett. 2010, 127, 85-92, the disclosure of which is incorporated herein by reference. IL-15 agonists are described, for example, in Wu, J Mol Genet Med. 2013, 7, 85 (the disclosure of which is incorporated herein by reference). In some embodiments, the IL-15R agonist may be a recombinant IL-15 molecule. In some embodiments, the IL-15R agonist may be a mimic of the IL-15 / IL-15Rα complex displayed on the cell surface, such as a heterodimeric complex or fusion protein comprising an IL-15 wild-type or mutant (e.g., N72D, D30N, E64Q, N65D) molecule and a partial or entire extracellular domain of IL-15Rα (e.g., soluble IL-15Rα), the sushi domain of IL-15Rα, or the like, optionally linked to one or more Fc domains. In some embodiments, the IL-15R agonist is a modified IL-15 molecule, such as an IL-15 mutant molecule (e.g., N72D, D30N, E64Q, N65D), an IL-15 with site-specific glycosylation(s), etc., which may have improved characteristics (e.g., increased half-life and increased affinity for IL-15R).

[0294] 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 by considering individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). 4 ~10 11 cells / kg body weight (e.g., 10 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 10 It may be specified that pharmaceutical compositions comprising tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein may be administered at dosages of 1000 to 10000 cells / kg body weight, including all integer values ​​within those ranges. TIL (including genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. TIL (including genetically engineered TILs in some cases) may be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg, et al., New Eng. J. of Med. 1988, 319, 1676). Optimal dosages and treatment regimes for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0295] The terms "hematological malignancy," "hematologic malignancy," or terms of related meaning refer to mammalian cancers and tumors of hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematologic malignancies are also referred to as "liquid tumors." Hematologic malignancies 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 hematologic malignancy" refers to a hematologic malignancy affecting B cells.

[0296] The term "liquid tumor" refers to an abnormal mass of cells that are liquid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors (including circulating liquid tumors 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 originate.

[0297] The term "microenvironment," as used herein, can refer to the solid tumor or blood tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanistic cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, promote therapeutic resistance, and provide a niche for advantageous metastasis and proliferation," 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.

[0298] In some embodiments, the invention encompasses methods of treating cancer with a population of TILs, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a population of TILs 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 at 60 mg / kg / day for two days (days 27 and 26 prior to TIL infusion) and fludarabine at 25 mg / m2 / day for five days (days 27-23 prior to TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (on day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours until physiological tolerance.

[0299] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in promoting 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 the introduction of the TILs of the present invention.

[0300] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended application, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the mode of administration. The term also applies to a dose that induces a particular response in target cells (e.g., reduced platelet adhesion and / or cell migration). Specific doses will vary depending on the particular compound selected, the dosing regimen 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 in which the compound is carried.

[0301] The terms "treatment," "treating," "treat," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in that the disease or condition is completely or partially prevented, and / or therapeutic, in that the disease and / or side effects resulting from the disease are partially or completely cured. "Treatment," as used herein, covers any treatment of disease in a mammal (especially a human), including (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting 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 cover the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" covers the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

[0302] As used herein, the term "immune checkpoint inhibitor (ICI)" has its general meaning in the art and refers to any compound that inhibits the function of an immune inhibitory checkpoint protein. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule expressed by T cells that either up-regulates a signal (a stimulatory checkpoint molecule) or down-regulates a signal (an inhibitory checkpoint molecule). Immune checkpoint molecules are recognized in the art to constitute elements of immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480-489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, CD277, IDO, KIR, VISTA, PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, Examples of immune checkpoint genes that can be silenced or inhibited in the TILs of the present invention include TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that can be silenced or inhibited in the TILs of the present invention can be selected from the group including PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018 (in press).According to another example, immune checkpoint genes that may be silenced or inhibited in the TILs of the present invention may be selected from the group including PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0303] Inhibition includes reduction and complete blockage of function. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. Many immune checkpoint inhibitors are known, and alternative immune checkpoint inhibitors that are similar to these known immune checkpoint protein inhibitors may be developed in the (near) future. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules, and small molecules.

[0304] 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 prior to administration of tumor-infiltrating lymphocytes to the patient as described herein.

[0305] 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. By way of example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes (e.g., a promoter from one source and a coding region from another source or coding regions from different sources) arranged to create a new functional nucleic acid. 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).

[0306] The terms "sequence identity," "percent identity," and "sequence percent identity" (or synonyms thereof, e.g., "99% identical"), in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or that have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering conservative amino acid substitutions as part of any 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 align amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST package of programs available from the U.S. government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTP is used to compare amino acid sequences, while BLASTN is used to compare nucleic 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 appropriate parameters for maximal alignment with a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0307] As used herein, the term "variant" encompasses, but is not limited to, antibodies or fusion proteins comprising an amino acid sequence that differs from that 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 the 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 encompasses PEGylated antibodies or proteins.

[0308] By "tumor infiltrating lymphocytes" or "TILs" herein is meant a population of cells originally 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 primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "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, second expanded TILs or second additional expanded TILs (e.g., including TILs referred to as reREP TILs, such as those described in step G of Figures 5A and 5C, step I of Figure 5B, and / or step H of Figure 5D).

[0309] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve 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 efficacy; for example, TILs can be determined to be efficacious if interferon (IFN) release is, for example, greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. TILs can be determined to be efficacious if interferon (IFNγ) release is, for example, 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.

[0310] 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 the oligonucleotide.

[0311] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide with a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA encompasses double-stranded RNA, single-stranded RNA, isolated RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification 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 modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0312] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to encompass 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 present invention is contemplated. Additional active pharmaceutical ingredients (such as other drugs) can also be incorporated into the compositions and methods described.

[0313] 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%, even 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 recognized 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 desired, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, as well as 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" regardless of whether it is explicitly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may use the described arrangements.

[0314] The transitional terms "comprising," "essentially consisting of," and "consisting of," when used in the appended claims, in their original and amended form, define the scope of the claims in terms of which additional unrecited claim elements or steps, if any, are excluded from the scope of the claim(s). The term "comprising" is intended to be inclusive or open-ended and does not exclude 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 impurities normally associated with the specified material(s) in the latter instance. 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 characteristic(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may be more specifically defined in alternative embodiments by any of the transitional terms "comprising," "essentially consisting of," and "consisting of."

[0315] 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 The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as V L The light chain constant region consists of one domain (C L ) V of the antibody H and V LThe regions of V can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs) or hypervariable regions (HVRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen epitope(s). 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 of the classical complement system (C1q).

[0316] 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 if presented by a major histocompatibility complex (MHC) molecule. The term "antigen," as used herein, 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 that leads to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be linked to a Th cell epitope. An antigen may also have one or more epitopes (e.g., a B epitope and a T epitope). In some embodiments, an antigen will preferably react with its corresponding antibody or TCR, typically in a highly specific and selective manner, and will not react with many other antibodies or TCRs that may be induced by other antigens.

[0317] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules in single molecular configuration. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be prepared using knowledge and skill in the art by injecting a suitable antigen into a test subject 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 capable of binding specifically 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 otherwise produce immunoglobulin proteins) to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.

[0318] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment"), as used herein, refers to one or more fragments 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) a Fab fragment (V L , V H , C L (ii) a F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region); (iii) a V H Domain and C H (iv) a single-arm V of an antibody; L and V H (v) an Fv fragment consisting of the domainsH or V L (vi) domain antibody (dAb) fragments, which may consist of two domains (V and V) of an Fv fragment (Ward, et al., Nature, 1989, 341, 544-546); and (vi) isolated complementarity-determining regions (CDRs). L and V H ) are encoded by separate genes, which can be engineered using recombinant methods to L Area and V H The domains may be linked by synthetic linkers, which allow them to be produced as a single protein chain, where they pair to form monovalent molecules known as single-chain Fvs (scFvs) (see, e.g., Bird, et al., Science 1988, 242, 423-426; and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the 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, an 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, then V L -LV H or V H If the domain is the N-terminal portion of the scFv molecule, then V H -LV LMethods for generating 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.

[0319] The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the present 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). The term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences.

[0320] 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.

[0321] The term "recombinant human antibody," as used herein, encompasses antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) 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, produced, or isolated by any other means involving 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. In certain embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H Area and V L The amino acid sequence of the region is human germline V H Sequence and V L These are sequences that, while derived from and related to a sequence, may not naturally exist within the human antibody germline repertoire in vivo.

[0322] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0323] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0324] 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.

[0325] The terms "humanized antibody(ies)" and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which residues from 15 hypervariable regions of a recipient (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one (typically two) variable domain, 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 optionally also 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 include, for example, those disclosed in International Patent Application Publication Nos. 1988 / 07089A1, 1996 / 14339A1, 1998 / 05787A1, 1998 / 23289A1, 1999 / 51642A1, 99 / 58572A1, 2000 / 09560A2, 2000 / 32767A1, 2000 / 42072A2, and 2002 / 44215A2; 2002 / 060919A2, 2003 / 074569A2, 2004 / 016750A2, 2004 / 029207A2, 2004 / 035752A2, 200 4 / 063351A2, 2004 / 074455A2, 2004 / 099249A2, 2005 / 040217A2, 2005 / 070963A1, 2005 / 077 981A2, 2005 / 092925A2, 2005 / 123780A2, 2006 / 019447A1, 2006 / 047350A2, and 2006 / 085967A2; and U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; Nos. 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).

[0326] 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 (such as 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).

[0327] A "diabody" is a small antibody fragment with two antigen-binding sites. The fragment contains a light chain variable domain (V) in the same polypeptide chain. L ) connected to a heavy chain variable domain (V H) including (VH-V L or V L -V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161; and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0328] The term "glycosylation" refers to a modified derivative of an antibody. A deglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the 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 elimination of one or more variable region framework glycosylation sites, thereby abolishing glycosylation at that site. Deglycosylation can increase the affinity of the antibody for an antigen, as described in U.S. Pat. Nos. 5,714,350 and 6,350,861. Additionally or alternatively, antibodies with altered types of glycosylation can be generated, 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 recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene (FUT8 (α(1,6)fucosyltransferase)), and as a result, antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrate. The FUT8- / - cell lines Ms704, Ms705, and Ms709 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; 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) such that antibodies expressed in the cell lines exhibit hypofucosylation due to reduced or absent α1,6 bond-related enzymes, and also describes cell lines (e.g., rat myeloma cell line YB2 / 0 (ATCC CRL 1662)) that have low or no enzymatic activity for adding fucose to N-acetylglucosamine attached to the Fc region of antibodies. International Patent Publication WO 03 / 035835 describes a variant CHO cell line (Lec 13 cells) that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication WO 99 / 54342 describes engineered cell lines that express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, the fucose residues of the antibody can be cleaved off using a fucosidase enzyme, for example, the fucosidase α-L-fucosidase, which removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0329] "PEGylation" typically 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 such that one or more PEG groups become 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 or alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to the form of PEG (mono(C1-C2)) that is used to derivatize other proteins. 10 ) alkoxy-polyethylene glycol or aryloxy-polyethylene glycol or polyethylene glycol-maleimide, etc. The PEGylated antibody may be a deglycosylated antibody. Methods for PEGylation are known in the art, 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), and can be applied to the antibodies of the present invention.

[0330] The term "biosimilar" refers to a biological product (including a monoclonal antibody or protein) that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, and that has no clinically meaningful differences between the biological product and the reference product with respect to product safety, purity, and potency. Furthermore, a similar biological drug or "biosimilar" drug is a biological drug that is similar to another biological drug already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by other national and regional regulatory agencies. Biological products or biological drugs are drugs 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, a protein approved by a drug regulatory agency with reference to aldesleukin is a "biosimilar" to aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological medicinal product, or "biosimilar" medicinal product, is a biological medicinal product that is similar to another biological medicinal product already approved for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, as amended. Therefore, in Europe, a biosimilar may be authorized, approved for authorization, or subject to authorization under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological medicinal product already authorized may be referred to in Europe as the "reference medicinal product." Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guidelines on similar biological medicinal products.In addition, product-specific guidelines (including guidelines related to monoclonal antibody biosimilars) are provided by the EMA on a product-by-product basis and published on its website. The biosimilars described herein may be similar to the reference pharmaceutical product in quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Additionally, the biosimilar may be used or intended for the treatment of the same condition as the reference pharmaceutical product. Thus, the biosimilars described herein may be considered to have similar or highly similar quality characteristics to the reference pharmaceutical product. Alternatively or additionally, the biosimilars described herein may be considered to have similar or highly similar biological activity to the reference pharmaceutical product. Alternatively or additionally, the biosimilars described herein may be considered to have similar or highly similar safety profile activity to the reference pharmaceutical product. Alternatively or additionally, the biosimilars described herein may be considered to have similar or highly similar efficacy to the reference pharmaceutical product. As described herein, biosimilars in Europe are compared to reference pharmaceutical products approved by the EMA. However, in some instances, a biosimilar may be compared in certain studies to a biological pharmaceutical product approved outside the European Economic Area (a non-EEA-approved "comparator"). Such studies include, for example, certain clinical studies and in vivo non-clinical studies. As used herein, the term "biosimilar" also relates to a biological pharmaceutical product that has been or can be compared to a non-EEA-approved comparator. Certain 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 in amino acid structure (e.g., 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 with 97% or greater (e.g., 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of its reference pharmaceutical product.A biosimilar may contain one or more post-translational modifications (e.g., but not limited to, glycosylation, oxidation, deamidation, and / or truncation) that differ from those of the reference pharmaceutical product, provided that these differences do not result in a change in the safety and / or efficacy of the pharmaceutical product. A biosimilar may have a glycosylation pattern that is identical to or different from that of the reference pharmaceutical product. In particular, but not limited to, a biosimilar may have a different glycosylation pattern if these differences address or are intended to address safety concerns associated with the reference pharmaceutical product. Additionally, a biosimilar may deviate from the reference pharmaceutical product, for example, in its strength, pharmaceutical form, formulation, excipients, and / or presentation, but the safety and efficacy of the pharmaceutical product are not compromised. A biosimilar may contain differences, for example, in its pharmacokinetic (PK) profile and / or pharmacodynamic (PD) profile compared to the reference pharmaceutical product, but still be considered sufficiently similar to the reference pharmaceutical product to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference pharmaceutical product, which are considered by regulatory authorities such as the EMA to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by other national and regional regulatory agencies.

[0331] The terms " organoid " and " tumor organoid " are used herein interchangeably and refer to patient-derived microspheres, which comprise dissociated primary tissue and cells (normal / healthy or abnormal / disease / cancerous) and optionally liquid matrix material, and the tissue and matrix material form unpolymerized tissue, which is then polymerized to form microspheres that are typically less than about 1000 μm in diameter.In some embodiments, organoids are less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, or less than 500 μm in diameter.

[0332] Dissociated primary tissue and / or cells may be freshly biopsied and obtained in any suitable manner, including mechanical or chemical dissociation (e.g., enzymatic disaggregation) through the use of one or more enzymes, such as collagenase, trypsin, etc. The dissociated tissue and / or cells may optionally be treated, selected, and / or modified. For example, cells may be sorted or selected to identify and / or isolate cells having one or more characteristics (e.g., size, morphology, etc.). Cells may be marked (e.g., with one or more markers) that can be used to aid selection. In some embodiments, cells may be sorted by well-characterized cell sorting techniques, including, but not limited to, microfluidic cell sorting, fluorescence-activated cell sorting, magnetic-activated cell sorting, etc.

[0333] The number of dissociated cells can be within the ranges mentioned above (e.g., about 1,000 to about 10,000 cells, about 10,000 to about 100,000 cells, about 100,000 to about 500,000 cells, about 500,000 to about 1,000,000 cells, about 1,000,000 to about 2,000,000 cells, or about 2,000,000 to about 3,000,000 cells). In some embodiments, one or more organoids or tumor organoids can contain about 3,000,000 tumor-derived cells. Any of these methods can be adapted as described herein to produce organoids or tumor organoids of reproducible size.

[0334] The process for producing organoids and tumor organoids has been well characterized and is disclosed in detail in at least the following patent applications: WO2019 / 067795 and US2021 / 0285054, which are incorporated by reference in their entireties.

[0335] II. Methods for Enriching Tumor-Reactive TILs Without being limited to any particular theory, it is believed that not all amplifiable TIL subpopulations have the same level of tumor reactivity, and that the tumor-reactive TIL subpopulations can be differentiated from these "bystander" TIL subpopulations through active selection based on phenotypic distinctions (such as IFNγ release or the protein expression profile of activation markers / exhaustion markers).It is also believed that such tumor-reactive TIL subpopulations can be enriched compared to the "bystander" TIL subpopulations by contacting them with autologous tumor digests or tumor lysates, by contacting them with mature dendritic cells that have previously been cultured with tumor antigens (in the form of tumor digests / tumor lysates or isolated peptides), or by contacting them with autologous tumor organoids or organoids.

[0336] Therefore, the present disclosure provides a method for enriching a plurality of tumor-reactive TILs. In some embodiments, the method comprises enriching tumor-reactive TILs before identifying a plurality of tumor-reactive TILs. In some embodiments, the enrichment step is performed after the first amplification step of TILs. In some embodiments, the enrichment step comprises: (a) co-culturing the TILs from the first amplification with autologous tumor digest or tumor lysate; (b) co-culturing the TILs from the first amplification with mature dendritic cells (which have previously been cultured with autologous tumor antigens in the form of tumor digest / tumor lysate or isolated peptides); or (c) co-culturing the TILs from the first amplification with autologous tumor organoids or organoids, so that the tumor-reactive TIL population is enriched. In some embodiments, the plurality of tumor-reactive TILs are then phenotypically profiled and / or identified. In some embodiments, the enriched plurality of tumor-reactive TILs are further amplified by a second amplification step. In some embodiments, the identified plurality of tumor-reactive TILs are further amplified by a second amplification step.

[0337] In some embodiments, profiling TILs to identify tumor-reactive subpopulations and isolating the tumor-reactive population are performed simultaneously, optionally using flow cytometry or other cell separation processes known to those skilled in the art. These processes include imaging-based methods that separate cell populations based on cell morphology. See generally Lin, W., et al. (2015). BMC Immunology, 16(1), 1-15. Examples of such imaging-based cell separation methods are disclosed in at least the following patent applications: WO2020 / 037070, US2021 / 0405022, US2021 / 0190669, and US2020 / 0150022, which are incorporated herein by reference in their entireties.

[0338] A. Obtaining Patient Tumor Samples Generally, TILs are initially obtained from patient tumor samples ("primary TILs") or from circulating lymphocytes (such as peripheral blood lymphocytes, including those with TIL-like characteristics), then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0339] Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, needle biopsy, or other means to obtain a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors (such as tumors obtained from hematological malignancies). Solid tumors can be any cancer type, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these have been reported to have particularly high levels of TILs.

[0340] Once obtained, tumor samples are generally extracted using sharp dissection to a size of 1 to about 8 mm. 3 fragmented into small pieces, especially about 2-3 mm 3is useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme-containing medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 micrograms / mL gentamicine, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme-containing medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for the methods of expanding TILs or treating cancer.

[0341] 1. Core / small biopsy-derived TILs In some embodiments, TILs are initially obtained from patient tumor samples obtained by core biopsy or similar procedures ("primary TILs"), then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, and optionally assessed for phenotypic and metabolic parameters.

[0342] In some embodiments, patient tumor samples can be obtained using methods known in the art, generally via mini-biopsy, core biopsy, needle biopsy, or other means to obtain a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors (such as tumors obtained from hematological malignancies). In some embodiments, samples can be from multiple small tumor samples or biopsies. In some embodiments, samples can include multiple tumor samples from a single tumor from the same patient. In some embodiments, samples can include multiple tumor samples from 1, 2, 3, or 4 tumors from the same patient. In some embodiments, samples can include multiple tumor samples from multiple tumors from the same patient. The solid tumor can be lung cancer and / or non-small cell lung cancer (NSCLC).

[0343] Generally, cell suspensions obtained from tumor cores or fragments are referred to as "primary cell populations" or "freshly obtained" or "freshly isolated" cell populations. In certain embodiments, freshly obtained cell populations of TILs are exposed to cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

[0344] In some embodiments, if the tumor is metastatic and the primary lesion has been effectively treated / removed, removal of one of the metastatic lesions may be necessary. In some embodiments, the least invasive approach is to remove the skin lesion or, if available, lymph nodes in the cervical or axillary region. In some embodiments, the skin lesion is removed, or a small biopsy thereof is removed. In some embodiments, a lymph node or a small biopsy thereof is removed. In some embodiments, the tumor is melanoma. In some embodiments, a small biopsy for melanoma includes a mole or a portion thereof.

[0345] In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin surrounding a suspicious mole. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin, removing a circular piece of skin. In some embodiments, the small biopsy is a punch biopsy, removing a circular portion of the tumor.

[0346] In some embodiments, the small biopsy is an excision biopsy. In some embodiments, the small biopsy is an excision biopsy, where the entire mole or growth is removed. In some embodiments, the small biopsy is an excision biopsy, where the entire mole or growth is removed along with a small border of normal-appearing skin.

[0347] In some embodiments, the mini-biopsy is an incisional biopsy. In some embodiments, the mini-biopsy is an incisional biopsy, where only the most irregular portion or growth of the mole is obtained. In some embodiments, the mini-biopsy is an incisional biopsy, where an incisional biopsy is used when other techniques cannot be completed (such as if the suspicious mole is very large).

[0348] In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Generally, with bronchoscopy, the patient is placed under anesthesia, and a small instrument is passed through the nose or mouth, down the throat, and into the bronchial passages, where the small instrument is used to remove some tissue. In some embodiments, if a tumor or growth cannot be reached through a bronchoscope, a transthoracic needle biopsy may be used. Generally, for a transthoracic needle biopsy, the patient is also under anesthesia, and a needle is inserted directly through the skin into the suspicious spot to remove a small sample of tissue. In some embodiments, a transthoracic needle biopsy may require interventional radiology (e.g., the use of an X-ray or CT scan to guide the needle). In some embodiments, the small biopsy is obtained by needle biopsy. In some embodiments, the small biopsy is obtained with an ultrasound endoscope (e.g., an endoscope with a light that is passed through the mouth and into the esophagus). In some embodiments, the small biopsy is obtained surgically.

[0349] In some embodiments, the minibiopsy is a head and neck biopsy. In some embodiments, the minibiopsy is an incisional biopsy. In some embodiments, the minibiopsy is an incisional biopsy, where a small piece of tissue is removed from an area that appears abnormal. In some embodiments, if the abnormal area is easily accessible, a sample can be obtained without hospitalization. In some embodiments, if the tumor is deep in the mouth or throat, the biopsy may need to be performed in an operating room under general anesthesia. In some embodiments, the minibiopsy is an excisional biopsy. In some embodiments, the minibiopsy is an excisional biopsy, where the entire area is removed. In some embodiments, the minibiopsy is a fine needle aspiration (FNA). In some embodiments, the minibiopsy is a fine needle aspiration (FNA), where a very thin needle attached to a syringe is used to extract (aspirate) cells from a tumor or lump. In some embodiments, the minibiopsy is a punch biopsy. In some embodiments, the minibiopsy is a punch biopsy, where a piece of suspicious area is removed using punch forceps.

[0350] In some embodiments, the mini-biopsy is a cervical biopsy. In some embodiments, the mini-biopsy is obtained via colposcopy. Generally, colposcopy involves the use of a lighted magnifying device attached to a magnifying binocular (colposcope), which is then used to biopsy a small section of the surface of the cervix. In some embodiments, the mini-biopsy is a cone biopsy. In some embodiments, the mini-biopsy is a cone biopsy, which may require outpatient surgery to remove a larger piece of tissue from the cervix. In some embodiments, a cone biopsy helps confirm a diagnosis, and in addition, a cone biopsy may serve as an initial treatment.

[0351] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells) and supportive stromal cells in which cancer cells may be dispersed and provide a supportive microenvironment.

[0352] In some embodiments, a sample from a tumor is obtained as a fine needle aspirate (FNA), core biopsy, or mini-biopsy (including, for example, a punch biopsy). In some embodiments, the sample is first placed into G-REX-10. In some embodiments, if there are one or two core biopsy and / or mini-biopsy samples, the sample is first placed into G-REX-10. In some embodiments, if there are three, four, five, six, eight, nine, or ten or more core biopsy and / or mini-biopsy samples, the sample is first placed into G-REX-100. In some embodiments, if there are three, four, five, six, eight, nine, or ten or more core biopsy and / or mini-biopsy samples, the sample is first placed into G-REX-500.

[0353] FNAs can be obtained from skin tumors, including, for example, melanoma. In some embodiments, FNAs are obtained from skin tumors, such as skin tumors from patients with metastatic melanoma. In some cases, melanoma patients have undergone previous surgical treatment.

[0354] FNAs can be obtained from lung tumors, including, for example, NSCLC. In some embodiments, FNAs are obtained from lung tumors, such as lung tumors from patients with non-small cell lung cancer (NSCLC). In some cases, patients with NSCLC have previously undergone surgical treatment.

[0355] The TIL described herein can be obtained from FNA sample.In some cases, FNA sample is obtained or isolated from patient using a thin gauge needle, ranging from 18 gauge needle to 25 gauge needle.The thin gauge needle can be 18 gauge, 19 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge, 24 gauge or 25 gauge. In some embodiments, an FNA sample from a patient may contain at least 400,000 TILs (e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more).

[0356] In some cases, the TIL described herein is obtained from core biopsy sample.In some cases, core biopsy sample is obtained or isolated from patient using surgical or medical needle ranging from 11 gauge needle to 16 gauge needle.Needle can be 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge or 16 gauge. In some embodiments, a core biopsy sample from a patient may contain at least 400,000 TILs (e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more).

[0357] Generally, the harvested cell suspension is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0358] 2. T cells and TILs in pleural effusions In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion according to the process described herein is a pleural fluid sample. In some embodiments, the sample is a pleural effusion-derived sample. In some embodiments, the source of T cells or TILs for expansion according to the process described herein is a pleural effusion-derived sample. For example, see the method described in US Patent Publication US2014 / 0295426 (incorporated herein by reference in its entirety for all purposes).

[0359] In some embodiments, any pleural fluid or pleural effusion suspected of containing and / or containing TILs can be used. Such samples can be derived from primary or metastatic lung cancer (such as NSCLC or SCLC). In some embodiments, the sample can be secondary metastatic cancer cells arising from another organ (e.g., breast, ovary, colon, prostate). In some embodiments, the sample for use in the amplification methods described herein is a pleural effusion. In some embodiments, the sample for use in the amplification methods described herein is a pleural transudate. Other biological samples can include other serous fluids containing TILs, including, for example, ascites from the abdomen or pancreatic cyst fluid. Ascites and pleural fluid contain very similar chemical systems; both the abdomen and lung have mesothelial lines and liquid morphology in the pleural and peritoneal cavities, similar to malignant tumors, and such fluids, in some embodiments, contain TILs. In some embodiments, while the present disclosure exemplifies pleural fluid, the same methods can be performed using ascites or other cyst fluids containing TILs, with similar results.

[0360] In some embodiments, the pleural fluid is in unprocessed form, as removed directly from the patient. In some embodiments, the unprocessed pleural fluid is placed in a standard blood collection tube (such as an EDTA or heparin tube) before the contacting step. In some embodiments, the unprocessed pleural fluid is placed in a standard CellSave® tube (Veridex) before the contacting step. In some embodiments, to avoid a decrease in the number of viable TILs, the sample is placed in a CellSave tube immediately after collection from the patient. The number of viable TILs can decrease significantly within 24 hours, even at 4°C, if left in unprocessed pleural fluid. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or a maximum of 24 hours after removal from the patient. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or a maximum of 24 hours at 4°C after removal from the patient.

[0361] In some embodiments, pleural fluid samples from selected subjects may be diluted. In some embodiments, the dilution is 1:10 pleural fluid to diluent. In other embodiments, the dilution is 1:9 pleural fluid to diluent. In other embodiments, the dilution is 1:8 pleural fluid to diluent. In other embodiments, the dilution is 1:5 pleural fluid to diluent. In other embodiments, the dilution is 1:2 pleural fluid to diluent. In other embodiments, the dilution is 1:1 pleural fluid to diluent. In some embodiments, the diluent includes saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, to avoid a significant loss of viable TILs within 24-48 hours if left in untreated pleural fluid, even at 4°C, the sample is placed in a CellSave tube immediately after collection from the patient and diluted. In some embodiments, the pleural fluid sample is placed in a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution. In some embodiments, the pleural fluid sample is placed in a suitable collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, or up to 48 hours after removal from the patient and dilution at 4°C.

[0362] In yet other embodiments, the pleural fluid sample is concentrated by conventional means prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in situations where the pleural fluid must be frozen for shipment to a laboratory performing the method or for later analysis (e.g., more than 24-48 hours after collection). In some embodiments, the pleural fluid sample is prepared by centrifuging the pleural fluid sample after withdrawal from the subject and resuspending the centrate or pellet in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugations and resuspensions, and then frozen for transport or later analysis and / or processing.

[0363] In some embodiments, the pleural fluid sample is concentrated by filtration before further processing. In some embodiments, the pleural fluid sample used in the contacting step is prepared by filtering the fluid through a filter containing a known, essentially uniform pore size that allows the passage of pleural fluid through the membrane but retains tumor cells. In some embodiments, the pore diameter in the membrane can be at least 4 μM. In other embodiments, the pore diameter can be 5 μM or greater, and in other embodiments, any of 6, 7, 8, 9, or 10 μM. After filtration, cells retained by the membrane (including TILs) can be rinsed from the membrane into a suitable physiologically acceptable buffer. Cells (including TILs) concentrated in this manner can then be used in the contacting step of the method.

[0364] In some embodiments, a pleural fluid sample (e.g., unprocessed pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lysis reagent that differentially lyses nonnucleated red blood cells present in the sample. In some embodiments, this step is performed in situations where the pleural fluid contains a substantial number of RBCs prior to further processing steps. Suitable lysis reagents include a single lysis reagent, a lysis reagent and a quenching reagent, or a lysis agent, a quenching reagent, and a fixation reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other lysis systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system, or the Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system. In some embodiments, the lytic reagent can vary depending on the primary requirements, which are efficient lysis of red blood cells and preservation of TILs and their phenotypic characteristics in the pleural fluid. In addition to using a single reagent for lysis, lytic systems useful in the methods described herein can include a second reagent (e.g., one that quenches or delays the effect of the lytic reagent during the remaining steps of the method, e.g., Stabilyse™ Reagent (Beckman Coulter, Inc.)). Conventional fixative reagents can also be used, depending on the choice of lytic reagent or the preferred implementation of the method.

[0365] In some embodiments, pleural fluid samples that have been unprocessed, diluted, or centrifuged or processed multiple times as described herein above are stored frozen at a temperature of about -140°C before being further processed and / or amplified as provided herein.

[0366] 3. Tumor Fragmentation and / or Digestion As described above, in some embodiments, the TILs are derived from a solid tumor. In some embodiments, when the tumor is a solid tumor, the tumor is subjected to physical fragmentation after a tumor sample is obtained, for example, in step A. In some embodiments, fragmentation is performed before cryopreservation. In some embodiments, fragmentation is performed after cryopreservation. In some embodiments, fragmentation is performed after obtaining the tumor and in the absence of any cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first amplification. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm. 3 In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments and has a volume of about 1300 mm 3 ~about 1500mm 3 In some embodiments, the plurality of pieces comprises about 50 pieces and has a total volume of about 1350 mm 3 In some embodiments, the plurality of pieces comprises about 50 pieces and has a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of pieces comprises about 4 pieces.

[0367] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are approximately 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor fragment is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor fragment is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor fragment is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor fragment is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor fragment is 4 mm x 4 mm x 4 mm.

[0368] In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue, necrotic tissue, and / or fatty tissue on each slice. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue on each slice. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each slice. In some embodiments, the tumor is fragmented to minimize the amount of fatty tissue on each slice. In certain embodiments, the step of fragmenting the tumor is an in vitro or ex vivo method.

[0369] In some embodiments, tumor fragmentation is performed to preserve the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without prior back-and-forth movement with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium (e.g., but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, and then mechanically disrupted again for approximately 1 minute. After again incubating at 37°C in 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large pieces of tissue were present after the third mechanical disruption, one or two additional rounds of mechanical dissociation were applied to the sample, with or without an additional 30-minute incubation at 37° C. in 5% CO. In some embodiments, if the cell suspension contained large numbers of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove these cells.

[0370] In some embodiments, the cell suspension prior to the first amplification step is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population.

[0371] In some embodiments, tumor lysates may be further obtained through multiple freeze-thaw cycles or mass spectrometry procedures from tumor digests.

[0372] In some embodiments, tumor fragments and / or tumor digests and / or tumor lysates may optionally be frozen and cryopreserved prior to entry into the first expansion step, TIL co-culture step, or DC pulsing step, as described in further detail below.

[0373] In some embodiments, the tumor is reconstituted with the lyophilized enzyme in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0374] In some embodiments, the enzyme mixture includes collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock for collagenase is a 10× working stock of 100 mg / mL.

[0375] In some embodiments, the enzyme mixture includes DNAse. In some embodiments, the working stock for DNAse is a 10× working stock of 10,000 IU / mL.

[0376] In some embodiments, the enzyme mixture includes hyaluronidase. In some embodiments, the working stock for hyaluronidase is a 10× working stock of 10 mg / mL.

[0377] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 1000 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0378] In some embodiments, the enzyme mixture comprises 10 mg / mL collagenase, 500 IU / mL DNAse, and 1 mg / mL hyaluronidase.

[0379] In some embodiments, fragmentation includes physical fragmentation (including, for example, dissection) and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.

[0380] In some embodiments, the TILs are not obtained from a tumor digest, hi some embodiments, the solid tumor core is not fragmented.

[0381] In some embodiments, obtaining the first population of TILs comprises a method of sampling multiple foci.

[0382] The tumor dissociation enzyme mixture may include one or more dissociating (digestive) enzymes, such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, protease type XIV (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, other dissociating or proteolytic enzymes, and any combination thereof.

[0383] In some embodiments, the dissociation enzyme is reconstituted from a lyophilized enzyme, hi some embodiments, the lyophilized enzyme is reconstituted in a volume of a sterile buffer, such as Hank's Balanced Saline Solution (HBSS).

[0384] In some instances, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer. Lyophilized stock enzyme may be at a concentration of 2892 PZ U / vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL of buffer. In some embodiments, after reconstitution, the collagenase stock has a concentration of about 100 U / mL to about 400 U / mL (e.g., about 100 U / mL to about 400 U / mL, about 100 U / mL to about 350 U / mL, about 100 U / mL to about 300 U / mL, about 150 U / mL to about 400 U / mL, about 100 U / mL, about 150 U / mL, about 200 U / mL, about 210 U / mL, about 220 U / mL, about 230 U / mL, about 240 U / mL, about 250 U / mL, about 260 U / mL, about 270 U / mL, about 280 U / mL, about 289.2 U / mL, about 300 U / mL, U / mL, about 350 PZ U / mL, or about 400 PZ U / mL).

[0385] In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme may be at a concentration of 175 DMC U / vial. In some embodiments, after reconstitution, the neutral protease stock may be at a concentration of about 100 DMC / mL to about 400 DMC / mL (e.g., about 100 DMC / mL to about 400 DMC / mL). DMC / mL, about 100 DMC / mL to about 350 DMC / mL, about 100 DMC / mL to about 300 DMC / mL, about 150 DMC / mL to about 400 DMC / mL, about 100 DMC / mL, about 110 DMC / mL, about 120 DMC / mL, about 130 DMC / mL, about 140 DMC / mL, about 150 DMC / mL, about 160 DMC / mL, about 170 DMC / mL, about 175 DMC / mL, about 180 DMC / mL, about 190 DMC / mL, about 200 DMC / mL, about 250 DMC / mL, about 300 DMC / mL, about 350 DMC / mL, or about 400 DMC / mL).

[0386] In some embodiments, DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer. The lyophilized stock enzyme was at a concentration of 4 KU / vial. In some embodiments, after reconstitution, the DNase I stock is in the range of about 1 KU / mL to 10 KU / mL (e.g., about 1 KU / mL, about 2 KU / mL, about 3 KU / mL, about 4 KU / mL, about 5 KU / mL, about 6 KU / mL, about 7 KU / mL, about 8 KU / mL, about 9 KU / mL, or about 10 KU / mL).

[0387] In some embodiments, enzyme stocks may vary, therefore verify the concentration of the lyophilized stock and adjust the final amount of enzyme added to the digestion cocktail accordingly.

[0388] In some embodiments, the enzyme mixture comprises about 10.2-ul neutral protease (0.36 DMC U / mL), 21.3-ul collagenase (1.2 PZ / mL), and 250-ul DNAse I (200 U / mL) in about 4.7 mL of sterile HBSS.

[0389] 4. Preparation of Crude Digests and Isolated Tumor Peptides In some embodiments, a portion of the tumor fragments is frozen and stored as a tumor cell suspension. In some embodiments, this suspension is thawed for use in a later step. In some embodiments, the thawed suspension is subjected to a dead cell removal kit before further use. In some embodiments, the thawed suspension is used without removing dead cells. In some embodiments, the tumor cell suspension is subjected to multiple freeze-thaw cycles. In different embodiments, the tumor cell suspension is subjected to 1, 2, 3, 4, 5, or 10 freeze-thaw cycles.

[0390] In other embodiments, the thawed suspension is further processed to produce tumor peptides, the process comprising homogenizing the tumor into small pieces, adding extraction buffer at a ratio of about 50:1, dissolving the resulting protein pellet in a volume of 8 M urea, 2 M soy urea, and 400 mM ammonium biocarbonate containing protease inhibitors, adding DTT, cooling and then heating the solution, and trypsinizing the solution.

[0391] 5. Generation of Mature Dendritic Cells In some embodiments, the methods disclosed herein involve generating mature dendritic cells (DCs) using the tumor digest or tumor lysate described herein. In some embodiments, the DCs are derived from peripheral blood monocytes. Means for generating mature DCs from monocytes are well known in the art. Briefly, peripheral blood mononuclear cells (PBMCs) from a cancer patient's apheresis or blood sample are incubated until the monocytes adhere to a substrate. The monocytes are cultured in cell culture medium containing GM-CSF and IL-4 for approximately 6 days to generate immature DCs. The immature DCs are then incubated with tumor digest or tumor lysate in cell culture medium containing TNFα, IL-6, and IL-1β to generate mature DCs. In some embodiments, the incubation lasts for 12 hours. In some embodiments, the incubation lasts for 16 hours. In some embodiments, the incubation lasts for 18 hours. In some embodiments, the incubation lasts for 24 hours. In some embodiments, the incubation lasts for 48 hours. In some embodiments, the incubation lasts for 72 hours. In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 10:1 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 5:1 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 3:1 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 2:1 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 1:1 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 1:2 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 1:3 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 1:5 (by number of cells). In some embodiments, the incubation comprises DC:tumor lysate at a ratio of about 1:10 (by number of cells).

[0392] 6. Generation of Organoids / Tumor Organoids Tumors possess certain physical characteristics that are difficult to mimic using traditional cell culture models (see generally Dao et al., Trends in Cancer, 2022, 8:10, pages 870-880). Therefore, in one aspect, the methods disclosed herein involve generating organoids and / or tumor organoids from tumor digests / tumor lysates for co-culturing with TILs after a first amplification step to enrich for tumor-reactive TILs. In some embodiments, organoids and / or tumor organoids are cultured from freshly biopsied primary tissue. In some embodiments, organoids and / or tumor organoids are cultured from cryopreserved primary tissue. In some embodiments, organoids and / or tumor organoids are cultured from one or more tumor fragments or tumor digests. In some embodiments, organoids and / or tumor organoids are cultured from one or more tumor fragments or tumor peptides. In some embodiments, organoids and / or tumor organoids are cultured from tumor-derived cells. In some embodiments, organoids and / or tumor organoids are cultured from a single donor. In some embodiments, organoids and / or tumor organoids are cultured from more than one donor. In some embodiments, organoids and / or tumor organoids are cultured for use in autologous therapy. In some embodiments, organoids and / or tumor organoids are cultured for use in allogeneic therapy. In some embodiments, organoids and / or tumor organoids comprise non-disease tissue. In some embodiments, organoids and / or tumor organoids comprise abnormal tissue or cancer tissue. In some embodiments, the tissue that becomes organoids and / or tumor organoids is cultured with a liquid matrix and manipulated to polymerize, and takes on an organ-like appearance.

[0393] In some embodiments, organoid or tumor organoid is cultured from fine needle aspirate.In some embodiments, FNA cells are cultured with Matrigel to promote organoid or tumor organoid formation.In some embodiments, stable cell line is generated from the cells of the formed organoid or tumor organoid.Additional experimental details of certain embodiments are described in Vilgelm, et al. (2020) iScience, 23(8), 101408 (the contents of which are incorporated herein by reference in their entirety).

[0394] In some embodiments, organoid and / or tumor organoid is useful for determining the tumor response resulting from a particular treatment.Additional experimental details of certain embodiments are described in Example 10 and in Dao et al., Trends in Cancer, 2022, 8:10, pages 870-880 (the contents of which are incorporated herein by reference in their entirety).

[0395] B. First Amplification In some embodiments, the methods disclosed herein provide tumor-reactive TILs, which may provide additional therapeutic benefit over bystander TILs (i.e., TILs that are capable of expansion but do not react to cancer tissue or cells). The dichotomy between tumor-reactive TILs and bystander TILs is described in the art in at least the following: Simoni, Y., et al. (2018). Nature, 557(7706), 575-579; Meier, SL, et al. (2022). Nature Cancer, 3(2), 143-155 (each of which is incorporated herein by reference).

[0396] After dissection of tumor tissue and / or tumor fragments, the resulting cells are cultured in serum containing IL-2 under conditions that favor the proliferation of TILs over tumor and other cells. In some embodiments, IL-2 is added at the initiation of culture (e.g., on day 0) along with tumor digests and / or tumor fragments. In some embodiments, tumor and / or tumor fragments are incubated in containers with up to 60 fragments per container and 6000 IU / mL of IL-2. In some embodiments, this primary cell population is cultured for a period of several days (generally 1-8 days) to produce a bulk TIL population (generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of several days (generally 1-7 days) to yield a bulk TIL population (generally about 1 x 10 8 In some embodiments, the first expansion is performed for a period of 1-8 days, resulting in a bulk TIL population (generally about 1 x 10 8 In some embodiments, the first expansion is performed for a period of 1-7 days, resulting in a bulk TIL population (generally about 1 x 10 8 In some embodiments, this first expansion is carried out for a period of 5-8 days, resulting in a bulk TIL population (generally about 1 x 10 8 In some embodiments, this first expansion is carried out for a period of 5-7 days, resulting in a bulk TIL population (generally about 1 x 10 8 In some embodiments, this first expansion is carried out for a period of about 6-8 days, resulting in a bulk TIL population (generally about 1 x 10 8 In some embodiments, this first expansion is carried out for a period of about 6-7 days, resulting in a bulk TIL population (generally about 1 x 10 8 In some embodiments, this first expansion is carried out for a period of about 7-8 days, resulting in a bulk TIL population (generally about 1 x 10 8In some embodiments, this first expansion is carried out for a period of about 7 days, resulting in a bulk TIL population (generally about 1 x 10 8 In some embodiments, this first expansion is carried out for a period of about 8 days, resulting in a bulk TIL population (generally about 1 x 10 8 resulting in a total of 100 TIL cells (bulk TIL cells).

[0397] In some embodiments, primary expansion of TILs can be accomplished using a process, which may include what is referred to as pre-REP or priming REP, which contains OKT-3 and feeder cells (e.g., antigen-presenting feeder cells) from day 0 and / or from the initiation of culture, as described below and herein, followed by rapid secondary expansion (Step G, which includes a process referred to as the Rapid Amplification Protocol (REP) step), as described below and herein, followed by optional cryopreservation. TILs obtained from this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some embodiments, tumor fragments are approximately 1 mm 3 ~10mm 3 is.

[0398] In some embodiments, the culture medium for the first amplification is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM for Step B consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin.

[0399] In some embodiments, there are 240 or fewer tumor fragments. In some embodiments, there are 240 or fewer tumor fragments placed in four or fewer containers. In some embodiments, the containers are GREX100 MCS flasks. In some embodiments, 60 or fewer tumor fragments are placed in one container. In some embodiments, each container contains 500 mL or fewer of medium per container. In some embodiments, the medium contains IL-2. In some embodiments, the medium contains 6000 IU / mL of IL-2. In some embodiments, the medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the medium contains 2.5 x 10 tumor fragments per container. 8 In some embodiments, the medium comprises OKT-3. In some embodiments, the medium comprises 30 ng / mL of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium comprises 6000 IU / mL of IL-2, 30 ng of OKT-3, and 2.5 x 10 8 In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 x 10 antigen-presenting feeder cells per vessel. 8 The antigen-presenting feeder cells are included.

[0400] After preparation of the tumor fragments, the resulting cells (i.e., the fragments are the primary cell population) are cultured in medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions that favor the proliferation of TILs over tumor and other cells, allowing for TIL priming and accelerated expansion starting from day 0 of culture. In some embodiments, tumor digests and / or tumor fragments are incubated with 6000 IU / mL of IL-2 and antigen-presenting feeder cells and OKT-3. This primary cell population is cultured for a period of several days (generally 1-8 days) to produce a bulk TIL population (generally about 1 x 10 8In some embodiments, the growth medium during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the growth medium during the first expansion further comprises antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for a period of several days (generally 1-7 days) to yield a bulk TIL population (generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion comprises IL-2 or a variant thereof and antigen-presenting feeder cells and OKT-3. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 In some embodiments, the IL-2 stock solution has a specific activity of 20×10 IU / mg per 1 mg vial. 6 In some embodiments, the IL-2 stock solution has a specific activity of 25×10 IU / mg per 1 mg vial. 6 In some embodiments, the IL-2 stock solution has a specific activity of 30×10 IU / mg per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution has a final concentration of 4-8 x 10 6 IU / mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 6 IU / mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×10 6 IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 4.

[0401] In some embodiments, the culture medium for the first amplification contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the culture medium for the first amplification contains about 9,000 IU / mL L-2 to about 5,000 IU / mL IL-2. In some embodiments, the culture medium for the first amplification contains about 8,000 IU / mL L-2 to about 6,000 IU / mL IL-2. In some embodiments, the culture medium for the first amplification contains about 7,000 IU / mL L-2 to about 6,000 IU / mL IL-2. In some embodiments, the culture medium for the first expansion comprises about 6,000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium for the first expansion comprises about 3,000 IU / mL of IL-2. In some embodiments, the cell culture medium for the first expansion further comprises IL-2. In some embodiments, the first cell culture medium comprises about 3,000 IU / mL of IL-2. In some embodiments, the cell culture medium for the first expansion contains about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium for the first amplification contains 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or about 8000 IU / mL of IL-2.

[0402] In some embodiments, the culture medium for the first expansion contains about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. In some embodiments, the culture medium for the first expansion contains about 500 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the culture medium for the first expansion contains about 400 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the culture medium for the first expansion contains about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the culture medium for the first expansion contains about 200 IU / mL of IL-15. In some embodiments, the cell culture medium for the first expansion contains about 180 IU / mL of IL-15. In some embodiments, the cell culture medium for the first expansion further contains IL-15. In some embodiments, the cell culture medium for the first expansion contains about 180 IU / mL of IL-15.

[0403] In some embodiments, the culture medium for the first expansion contains about 20 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the culture medium for the first expansion contains about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the culture medium for the first expansion contains about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the culture medium for the first expansion contains about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the culture medium for the first expansion contains about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the culture medium for the first expansion contains about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the culture medium for the first expansion contains about 2 IU / mL of IL-21. In some embodiments, the cell culture medium for the first expansion contains about 1 IU / mL of IL-21. In some embodiments, the cell culture medium for the first expansion contains about 0.5 IU / mL of IL-21. In some embodiments, the cell culture medium further contains IL-21. In some embodiments, the cell culture medium for the first expansion contains about 1 IU / mL of IL-21.

[0404] In some embodiments, the cell culture medium for the first amplification comprises an OKT-3 antibody. In some embodiments, the cell culture medium for the first amplification comprises about 30 ng / mL of the OKT-3 antibody. In some embodiments, the cell culture medium for the first amplification comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In some embodiments, the cell culture medium comprises 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 15 ng / mL to 30 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises 30 ng / mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab. See, e.g., Table 1.

[0405] In some embodiments, the cell culture medium for the first amplification comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL in the cell culture medium.

[0406] In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium for the first expansion further comprises IL-2 at an initial concentration of about 3000 IU / mL and an OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to the one or more TNFRSF agonists, the cell culture medium for the first expansion further comprises IL-2 at an initial concentration of about 6000 IU / mL and an OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0407] In some embodiments, the culture medium for the first expansion is referred to as "CM" (abbreviation for culture medium). In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM1 consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In some embodiments, the CM is CM1 described in the Examples. In some embodiments, the first expansion is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the first expansion culture medium or the initial cell culture medium or the first cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells (also referred to herein as feeder cells).

[0408] In some embodiments, the culture medium used in the expansion processes disclosed herein is a serum-free or synthetic medium. In some embodiments, the serum-free or synthetic medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the serum-free or synthetic medium is used to prevent and / or reduce experimental variation due in part to lot-to-lot variation of serum-containing medium.

[0409] In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium (CTS™ OpTmizer™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0410] In some embodiments, serum supplements or serum replacements include, but are not limited to, one or more of CTS™ OpTmizer T-Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, a synthetic medium contains albumin, as well as glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element moieties. + , Al 3+ , Ba 2+ , Cd 2+, Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ In some embodiments, the synthetic medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0411] In some embodiments, CTS™ OpTmizer™ T-cell Immune Cell Serum Replacement is used in conjunction with conventional growth media, including, but not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0412] In some embodiments, the total serum replacement concentration (volume %) in the serum-free or synthetic medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the complete serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or synthetic medium.

[0413] In some embodiments, the serum-free or synthetic medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, mixed together prior to use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0414] In some embodiments, the defined medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, mixed together prior to use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 3000 IU / mL IL-2.In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 1000 IU / mL to about 8000 IU / mL of IL-2.In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU / mL IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU / mL IL-2. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0415] In some embodiments, the serum-free or synthetic medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free or synthetic medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0416] In some embodiments, the serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0417] In some embodiments, the synthetic media described in International PCT Publication No. 1998 / 030679 (which is incorporated herein by reference) are useful in the present invention. Serum-free eukaryotic cell culture media are described in that publication. The serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement includes, or is obtained by combining, one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the synthetic media further include L-glutamine, sodium bicarbonate, and / or β-mercaptoethanol. In some embodiments, the synthetic medium comprises albumin or an albumin substitute, and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the synthetic medium comprises albumin, and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ , Se 4+, Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0418] In some embodiments, the concentration of glycine in the synthetic medium ranges from about 5 to 200 mg / L, the concentration of L-histidine from about 5 to 250 mg / L, the concentration of L-isoleucine from about 5 to 300 mg / L, the concentration of L-methionine from about 5 to 200 mg / L, the concentration of L-phenylalanine from about 5 to 400 mg / L, the concentration of L-proline from about 1 to 1000 mg / L, the concentration of L-hydroxyproline from about 1 to 45 mg / L, the concentration of L-serine from about 1 to 250 mg / L, the concentration of L-threonine from about 10 to 500 mg / L, and the concentration of L-tryptophan from about 2 to 110 mg / L. / L, the concentration of L-tyrosine is about 3-175 mg / L, the concentration of L-valine is about 5-500 mg / L, the concentration of thiamine is about 1-20 mg / L, the concentration of reduced glutathione is about 1-20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1-200 mg / L, the concentration of iron-saturated transferrin is about 1-50 mg / L, the concentration of insulin is about 1-100 mg / L, the concentration of sodium selenite is about 0.000001-0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5000-50,000 mg / L.

[0419] In some embodiments, non-trace element components in the defined medium are present at the concentration ranges listed in the column under the heading "Concentration Ranges in 1x Medium" in Table 5. In other embodiments, non-trace element components in the defined medium are present at the final concentrations listed in the column under the heading "Preferred Embodiment in 1x Medium" in Table 5. In other embodiments, the defined medium is a basal cell culture medium that includes a serum-free supplement. In some of these embodiments, the serum-free supplement includes non-trace element components of the type and concentration listed in Table 5 in the column under the heading "Preferred Embodiment in Supplement."

[0420] [Table 4]

[0421] In some embodiments, the osmolality of the synthetic medium is about 260-350 mOsmol. In some embodiments, the osmolality is about 280-310 mOsmol. In some embodiments, the synthetic medium is supplemented with up to about 3.7 g / L or about 2.2 g / L sodium bicarbonate. The synthetic medium may be further supplemented with L-glutamine (about 2 mM final concentration), one or more antibiotics, non-essential amino acids (NEAA; about 100 μM final concentration), and 2-mercaptoethanol (about 100 μM final concentration).

[0422] In some embodiments, the synthetic media described in Smith, et al., Clin. Transl. Immunology, 4(1), 2015 (doi:10.1038 / cti.2014.31) are useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ is used as the basal cell culture medium, supplemented with either 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0423] In some embodiments, the cell culture medium in the first and / or second gas permeable container is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand cell numbers. In some embodiments, the cell culture medium in the first and / or second gas permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0424] In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 1 to 11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 2 to 11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 3 to 11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 4 to 11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 5 to 11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 6 to 11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 7-11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 8-11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 9-11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 10-11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 11 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 1-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming REP) is about 2-10 days.In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 3-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 4-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 5-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 6-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 7-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 8-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 9-10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 10 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 1-9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 2-9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 3-9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 4-9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 5-9 days.In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 6-9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 7-9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 8-9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 9 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 1-8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 2-8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) takes about 3-8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 4-8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 5-8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 6-8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 7-8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 8 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 1-7 days.In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 2-7 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 3-7 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 4-7 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 5-7 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 6-7 days. In some embodiments, the first amplification process (including processes such as those sometimes referred to as pre-REP or priming-REP) is about 7 days.

[0425] In some embodiments, the first TIL amplification may proceed for 1 to 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 1 to 7 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 2 to 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 2 to 7 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 3 to 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 3 to 7 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 4 to 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 4 to 7 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 5 to 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 5 to 7 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 6 to 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 6 to 7 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 7 to 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated. In some embodiments, the first TIL amplification may proceed for 8 days from the time when fragmentation is performed and / or the time when the first amplification step is initiated.In some embodiments, the first TIL amplification is allowed to proceed for 7 days from the time fragmentation occurs and / or the first amplification step is initiated.

[0426] In some embodiments, the first TIL amplification may proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days. In some embodiments, the first TIL amplification may proceed for 1 to 9 days. In some embodiments, the first TIL amplification may proceed for 1 to 8 days. In some embodiments, the first TIL amplification may proceed for 1 to 7 days. In some embodiments, the first TIL amplification may proceed for 2 to 9 days. In some embodiments, the first TIL amplification may proceed for 2 to 8 days. In some embodiments, the first TIL amplification may proceed for 2 to 7 days. In some embodiments, the first TIL amplification may proceed for 3 to 9 days. In some embodiments, the first TIL amplification may proceed for 3 to 8 days. In some embodiments, the first TIL amplification may proceed for 3 to 7 days. In some embodiments, the first TIL amplification may proceed for 4 to 9 days. In some embodiments, the first TIL amplification may proceed for 4 to 8 days. In some embodiments, the first TIL amplification may proceed for 4 to 7 days. In some embodiments, the first TIL amplification may proceed for 5 to 9 days. In some embodiments, the first TIL amplification may proceed for 5 to 8 days. In some embodiments, the first TIL amplification may proceed for 5 to 7 days. In some embodiments, the first TIL amplification may proceed for 6 to 9 days. In some embodiments, the first TIL amplification may proceed for 6 to 8 days. In some embodiments, the first TIL amplification may proceed for 6 to 7 days. In some embodiments, the first TIL amplification may proceed for 7 to 9 days. In some embodiments, the first TIL amplification may proceed for 7 to 8 days. In some embodiments, the first TIL amplification may proceed for 8 to 9 days. In some embodiments, the first TIL amplification may proceed for 9 days. In some embodiments, the first TIL amplification may proceed for 8 days. In some embodiments, the first TIL amplification may proceed for 7 days. In some embodiments, the first TIL amplification may proceed for 6 days.

[0427] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-2 is used as a combination during priming in the first amplification. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, can be included during the first amplification. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during priming in the first amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, can be included during the first amplification.

[0428] In some embodiments, the first amplification is carried out in a closed system bioreactor. In some embodiments, a closed system is used for TIL amplification as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as a vessel. In some embodiments, the bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-10.

[0429] 1. Feeder cells and antigen-presenting cells In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time between days 4 and 8 during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time between days 4 and 7 during the first amplification. In some embodiments, the first amplification priming procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather they are added any time between days 5 and 8 during the priming amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather they are added any time between days 5 and 7 during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather they are added any time between days 6 and 8 during the first amplification.In some embodiments, the first amplification priming procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time between days 6 and 7 during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time between days 7 and 8 during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time between days 6 and 7 during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time up to day 7 during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time up to day 8 during the first amplification. In some embodiments, the first amplification procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL amplification, but rather, they are added any time up to day 9 during the first amplification.

[0430] In some embodiments, the first expansion procedure described herein (including, for example, amplifications such as those referred to as pre-REP or priming-REP) requires feeder cells (also referred to herein as "antigen-presenting cells") at the beginning of TIL expansion and during the first expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic healthy blood donor. PBMCs are obtained using standard methods (such as Ficoll-Paque gradient separation). In some embodiments, 2.5 x 10 8 In some embodiments, 2.5 x 10 feeder cells per vessel are used during the first amplification. 8 In some embodiments, 2.5 x 10 feeder cells per GREX-10 are used during the first amplification. 8 In some embodiments, 2.5 x 10 feeder cells per GREX-100 are used during the first amplification. 8 of feeder cells are used during the first amplification.

[0431] Generally, allogeneic PBMCs are inactivated via either irradiation or heat treatment and used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessment of the replication incompetence of irradiated allogeneic PBMCs.

[0432] In some embodiments, PBMCs are deemed non-replicating and acceptable for use in the TIL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial number of viable cells placed into culture on day 0 of the first expansion.

[0433] In some embodiments, PBMCs are deemed replication-incompetent and acceptable for use in the TIL expansion procedures described herein if the total number of viable cells on day 7 cultured in the presence of OKT3 and IL-2 has not increased from the initial number of viable cells placed into culture on day 0 of the first expansion. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 6000 IU / mL IL-2.

[0434] In some embodiments, PBMCs are deemed replication-incompetent and acceptable for use in the TIL expansion procedures described herein if the total number of viable cells on day 7 of culture in the presence of OKT3 and IL-2 has not increased from the initial number of viable cells placed into culture on day 0 of the first expansion. In some embodiments, PBMCs are cultured in the presence of 5-60 ng / mL OKT3 antibody and 1000-6000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng / mL OKT3 antibody and 2500-3500 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 6000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng / mL OKT3 antibody and 3000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng / mL OKT3 antibody and 6000 IU / mL IL-2.

[0435] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second amplification is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second amplification is 1:50 to 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second amplification is 1:100 to 1:200.

[0436] In some embodiments, the first amplification procedure described herein produces approximately 2.5 x 10 8 of feeder cells versus approximately 100 x 10 6 In other embodiments, the first amplification procedure described herein requires a ratio of about 2.5 x 10 TILs. 8 of feeder cells versus approximately 50 x 10 6 In yet another embodiment, the first amplification described herein requires a ratio of about 2.5 x 10 TILs. 8 of feeder cells versus approximately 25 x 10 6 In yet another embodiment, the first amplification described herein requires about 2.5 x 10 TILs. 8 In still other embodiments, the first amplification requires one-quarter, one-third, five-twelfths, or one-half the number of feeder cells used in the rapid second amplification.

[0437] In some embodiments, the medium for the first amplification contains IL-2. In some embodiments, the medium for the first amplification contains 6000 IU / mL of IL-2. In some embodiments, the medium for the first amplification contains antigen-presenting feeder cells. In some embodiments, the medium for the first amplification contains 2.5 x 10 cells per container. 8 In some embodiments, the medium in the first expansion comprises OKT-3. In some embodiments, the medium comprises 30 ng of OKT-3 per vessel. In some embodiments, the vessel is a GREX100 MCS flask. In some embodiments, the medium comprises 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 x 10 8 In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5 x 10 antigen-presenting feeder cells per vessel. 8 In some embodiments, the medium comprises 500 mL of culture medium and 2.5 x 10 antigen-presenting feeder cells per vessel. 8 In some embodiments, the medium comprises 500 mL of culture medium and 15 μg of OKT-3 per vessel. In some embodiments, the vessel is a GREX100 MCS flask. In some embodiments, the medium comprises 500 mL of culture medium, 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 In some embodiments, the medium contains 500 mL of culture medium, 6000 IU / mL of IL-2, 15 μg of OKT-3, and 2.5×10 antigen-presenting feeder cells per container. 8 In some embodiments, the medium comprises 500 mL of culture medium and 2.5 x 10 antigen-presenting feeder cells per vessel. 8 Contains 15 μg of OKT-3 per antigen-presenting feeder cell.

[0438] In some embodiments, the first expansion procedure described herein requires an excess of feeder cells over TILs during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic healthy blood donor. PBMCs are obtained using standard methods (such as Ficoll-Paque gradient separation). In some embodiments, artificial antigen-presenting (aAPC) cells are used instead of PBMCs.

[0439] Generally, allogeneic PBMCs are inactivated via either irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.

[0440] In some embodiments, artificial antigen-presenting cells are used in the first expansion, either as a replacement for or in combination with PBMCs.

[0441] 2. Cytokines and other additives The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as is known in the art.

[0442] Alternatively, the use of a combination of cytokines for the initial expansion of TILs is additionally possible using a combination of two or more of IL-2, IL-15, and IL-21, as described in U.S. Patent Application Publication No. 2017 / 0107490A1 (the disclosure of which is incorporated herein by reference). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter of which finds particular use in many embodiments. The use of a combination of cytokines specifically favors the generation of lymphocytes (particularly T cells as described herein). See, for example, Table 2.

[0443] In some embodiments, step B may also include the addition of an OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may also include the addition of an OX-40 agonist to the culture medium, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator Iα agonists, including proliferator-activated receptor (PPAR)-γ agonists such as thiazolidinedione compounds, may be used in the culture medium during step B, as described in U.S. Patent Application Publication No. 2019 / 0307796A1 (the disclosure of which is incorporated herein by reference).

[0444] C. Enrichment of tumor-reactive TILs In some embodiments, the methods disclosed herein include enriching tumor-reactive TILs, e.g., after a first amplification step. In some embodiments, prior to the enrichment step, residual tumor fragments are removed from the TILs after the first amplification step.

[0445] In some embodiments, the enrichment step includes (a) co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate; (b) co-culturing the TILs from the first expansion with mature dendritic cells (which have previously been cultured with autologous tumor antigens in the form of either tumor digest / tumor lysate or isolated peptides); or (c) co-culturing the TILs from the first expansion with autologous tumor organoids or organoids, so as to enrich the tumor-reactive TIL population.

[0446] In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 1:10. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 1:5. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 1:3. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 1:2. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 1:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 2:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 3:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 5:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor digest or tumor lysate at a TIL:tumor cell ratio of 10:1.

[0447] In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 1:10. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 1:5. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 1:3. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 1:2. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 1:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 2:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 3:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 5:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with mature DCs at a TIL:DC ratio of 10:1.

[0448] In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 1:10. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 1:5. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 1:3. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 1:2. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 1:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 2:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 3:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 5:1. In some embodiments, the enrichment step comprises co-culturing the TILs from the first expansion step with autologous tumor organoids or organoids at a TIL:tumor cell ratio of 10:1.

[0449] In some embodiments, the enrichment step is carried out for about 12 hours, about 16 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0450] In some embodiments, the tumor reactivity of TILs after the enrichment step can be assessed by measuring one or more characteristics of the TILs. For example, one or more secretory factors (e.g., presence or absence, concentration, specific activity) in the TIL cell culture supernatant can be measured. In some embodiments, the measurable secretory factor is one or more cytokines. In some embodiments, the measurable secretory factor is IFN-γ.

[0451] D. Identification of Multiple Tumor-Reactive TILs In some embodiments, the methods disclosed herein further include identifying multiple tumor-reactive TILs, which can be collected and further expanded as disclosed herein.

[0452] In some embodiments, identifying multiple tumor-reactive TILs may include determining whether the TILs exhibit activation signals that identify the TILs as tumor-reactive (such as changes in cell morphology (e.g., immunological synapse formation and cell shape), changes in cell surface expression of one or more proteins, changes in secretion levels of one or more cytokines, changes in mRNA expression, etc.).

[0453] In some embodiments, the activation signal may comprise an increase and / or decrease in cell surface expression of one or more proteins. In some embodiments, the one or more proteins comprise a T cell activation or exhaustion marker. In some embodiments, the T cell activation or exhaustion marker is selected from the group consisting of CD3, CD4, CD8, PD-1, LAG3, Tim3, TIGIT, CD103, CD39, CD134, CD137, CD25, CD69, HLA-DR, CD107A, CD40L, Ki67, CD45RA, CCR7, KLRG1, and combinations thereof.

[0454] In some embodiments, the T cell activation or exhaustion marker is a cell surface protein. In some embodiments, determining whether the TILs exhibit an activation signal is accomplished by staining the population of TILs after the enrichment step with an antibody against a T cell activation or exhaustion marker. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody.

[0455] In some embodiments, the activation signal can include cell morphology. In some embodiments, the cell morphology is a flat, rounded cell morphology. See Lin, et al., BMC Immunol. 2015, 16, 49, the contents of which are incorporated herein by reference in their entireties. In some embodiments, the activation signal is the concentration of mitochondrial mass adjacent to the cell membrane of TILs. In some embodiments, the cell morphology is determined by an imaging-based cell separation method. In some embodiments, the imaging-based cell separation method uses a cell sorting system, such as the methods and cell sorting systems described in WO2020037070A1 and US2021 / 0190669A1, the contents of which are incorporated herein by reference in their entireties.

[0456] In some embodiments, the methods disclosed herein further include collecting a plurality of tumor-reactive TILs. In some embodiments, collecting a plurality of tumor-reactive TILs includes separating the plurality of tumor-reactive TILs from non-tumor-reactive TILs. In some embodiments, the collection is accomplished using a cell sorting method. In some embodiments, the cell sorting method is a flow cytometry method, such as flow-activated cell sorting (FACS). In some embodiments, gating is set for each sort. In some embodiments, gating is set for each TIL sample. In some embodiments, a gating template is set from the TILs every 10, 20, 30, 40, 50, or 60 days. In some embodiments, a gating template is set from the TILs every 60 days. In some embodiments, a gating template is set for each TIL sample every 10, 20, 30, 40, 50, or 60 days. In some embodiments, a gating template is set for each PBMC sample every 60 days. In some embodiments, flow cytometry is performed using a SONY FX 500, Miltenyi Tyto, or Miltenyi CliniMACS flow activated cell sorter.

[0457] In some embodiments, the collecting is accomplished using an imaging-based cell sorting method. In some embodiments, the method further includes (i) providing a cell population containing cells, (ii) analyzing a subpopulation of the cell population at a first time to detect target cells, (iii) collecting the subpopulation if the number of target cells in the subpopulation exceeds a predetermined threshold number, and (iv) analyzing the subpopulation at a second time. In some embodiments, the method further includes, in (ii), capturing one or more images of each cell in the subpopulation. In some embodiments, the method further includes capturing a single image of each cell from a single angle.

[0458] In some embodiments, the method further includes (a) transporting cells through a flow channel; (b) capturing multiple images of the cells from multiple different angles as the cells are transported through the flow channel; and (c) analyzing the multiple images using a deep learning algorithm to sort the cells. In some embodiments, the method further includes rotating the cells as they are transported through the flow channel. In some embodiments, the method further includes applying a velocity gradient across the cells to rotate the cells. In some embodiments, the cells are flowed in a first buffer at a first velocity, and applying the velocity gradient across the cells includes co-flowing a second buffer at a second velocity. In some embodiments, the axis of rotation of the cells and the additional axis of movement of the cells along the flow channel are different. In some embodiments, the axis of rotation of the cells is perpendicular to the additional axis of movement of the cells along the flow channel. In some embodiments, the method further includes focusing the cells into streamlines at a height within the flow channel as the cells are transported through the flow channel. In some embodiments, the focusing includes subjecting the cells to an inertial lift force, the inertial lift force characterized by a Reynolds number greater than 1. In some embodiments, the inertial lift force is characterized by a Reynolds number of at least 20. In some embodiments, the multiple images are captured at a rate of about 10 frames / second to about 500,000 frames / second. In some embodiments, the multiple angles extend around or across a portion of the cell. In some embodiments, the multiple images of the cell are captured from (1) the top side of the cell, (2) the bottom side of the cell, (3) the front side of the cell, (4) the back side of the cell, (5) the left side of the cell, or (6) the right side of the cell. In some embodiments, the multiple images of the cell are captured from at least two sides selected from the group consisting of (1) the top side of the cell, (2) the bottom side of the cell, (3) the front side of the cell, (4) the back side of the cell, (5) the left side of the cell, and (6) the right side of the cell.In some embodiments, the method further includes sorting the cells based on the analyzed images by directing the cells to a selected channel of a plurality of channels downstream of the flow channel, hi some embodiments, the plurality of channels except for the selected channel are blocked prior to directing the cells to the selected channel.

[0459] In some embodiments, the plurality of channels, except for the selected channel, are blocked using pressure, an electric field, a magnetic field, or a combination thereof. In some embodiments, the method further includes verifying the sorting of the cells using illumination. In some embodiments, the verification includes determining information associated with the cells using blocking or scattering of illumination. In some embodiments, the information associated with the cells includes cell size, shape, density, texture, or velocity. In some embodiments, the verification includes (i) providing at least two illumination spots on the selected channel by directing at least two illumination spots onto the selected channel, and (ii) determining the cell's transit time between the at least two illumination spots, the at least two illumination spots being spaced about 10 micrometers to about 1,000 micrometers apart. In some embodiments, the illumination includes a laser. In some embodiments, the sorting includes (i) directing a first cell into a first channel of the plurality of channels and (ii) directing a second cell into a second channel of the plurality of channels, wherein the first cell and the second cell have (or are suspected of having) one or more distinct characteristics.

[0460] In some embodiments, the method further includes sorting the plurality of cells at a rate of at least 10 cells / second, where the plurality of cells comprises a single cell. In some embodiments, the method further includes using a classifier to sort the plurality of cells, including the single cell; and feeding data from the sort back to the classifier to train the classifier for future sorts. In some embodiments, the classifier comprises a neural network. In some embodiments, the classifier is configured to perform classification of each of the plurality of cells based on classification probabilities corresponding to a plurality of analyzed images of the plurality of cells.

[0461] In some embodiments, the method further includes (a) obtaining spatial information during movement of the plurality of cells relative to the patterned optical structure; (b) using the spatial information to identify one or more target cells from the plurality of cells; and (c) separating or isolating one or more target cells from the plurality of cells at a rate of at least 10 cells / second based at least in part on the one or more target cells identified in (b). In some embodiments, (a) includes (i) directing illumination from a light source through the patterned optical structure, (ii) directing illumination from the patterned optical structure to the plurality of cells, and (iii) directing illumination from the plurality of cells to a detector. In some embodiments, (a) includes (i) directing illumination from a light source to the plurality of cells, (ii) directing illumination from the plurality of cells through the patterned optical structure, and (iii) directing illumination from the patterned optical structure to a detector. In some embodiments, the patterned optical structure includes a non-regularly patterned optical structure. In some embodiments, (c) includes computationally reconstructing the cellular morphology at least in part through the use of a combination of one or more temporal waveforms comprising one or more intensity distributions imparted by the patterned optical structure. In some embodiments, the target cells are tumor-reactive TILs. In some embodiments, (b) includes applying one or more machine learning classifiers to the compressed waveforms corresponding to the spatial information to identify the one or more target cells. In some embodiments, the one or more machine learning classifiers achieve one or more of a sensitivity, specificity, and accuracy of at least 70%. In some embodiments, the one or more machine learning classifiers are selected from the group consisting of support vector machines, random forests, artificial neural networks, convolutional neural networks, deep learning, very deep learning, gradient boosting, AdaBoosting, decision trees, linear regression, and logistic regression. In some embodiments, the plurality of cells is processed without image reconstruction.In some embodiments, the detector comprises a single-pixel detector. In some embodiments, the single-pixel detector comprises a photomultiplier tube. In some embodiments, the method further comprises reconstructing one or more images of the plurality of cells. In some embodiments, the method further comprises reconstructing multiple images of the plurality of cells, each of the plurality of images comprising a different wavelength or wavelength range. In some embodiments, the one or more images are free of blurring artifacts. In some embodiments, the plurality of cells move at a speed of at least 1 m / s relative to the patterned optical structure. In some embodiments, (c) comprises (i) sorting the plurality of cells into one or more groups of sorted cells based on an analysis result of the plurality of cells; and (ii) collecting one or more target cells from the one or more groups of sorted cells. In some embodiments, (c) comprises sorting the plurality of cells into one or more groups of sorted cells based on a morphology of the plurality of cells. In some embodiments, the sorting is accomplished at a speed of at least 10 cells / s. In some embodiments, the method further comprises collecting one or more of the groups of sorted cells to generate an enriched cell mixture. In some embodiments, the one or more groups of sorted cells have a purity of at least 70%. In some embodiments, the method further comprises subjecting one or more cells of the one or more groups of sorted cells to one or more assays. In some embodiments, the one or more assays are selected from the group consisting of lysis, nucleic acid extraction, nucleic acid amplification, nucleic acid sequencing, and protein sequencing. In some embodiments, the method further comprises, prior to (a), subjecting the cells to hydrodynamic flow focusing. In some embodiments, the method further comprises collecting a speckle pattern of partial transmission of the plurality of cells as the plurality of cells move relative to the patterned optical structure. In some embodiments, the spatial information corresponds to features, characteristics, or information belonging to the plurality of cells.In some embodiments, the spatial information corresponds one-to-one with features, characteristics, or information belonging to the plurality of cells. In some embodiments, the features, characteristics, or information belonging to the plurality of cells comprises one or more members selected from the group consisting of metabolic state, proliferation state, differentiation state, maturation state, expression of a marker protein, expression of a marker gene, cell morphology, organelle morphology, organelle location, organelle size or extent, cytoplasmic morphology, cytoplasmic location, cytoplasmic size or extent, nuclear morphology, nucleus location, nucleus size or extent, mitochondrial morphology, mitochondrial location, mitochondrial size or extent, lysosomal morphology, lysozyme location, lysozyme size or extent, distribution of molecules within the cell, distribution of peptides, polypeptides, or proteins within the cell, distribution of nucleic acids within the cell, distribution of glycans or polysaccharides within the cell, and distribution of lipids within the cell.

[0462] In some embodiments, the TILs are profiled before being subjected to the imaging-based cell sorting method, while in other embodiments, the TILs are directly subjected to the imaging-based cell sorting method without profiling.

[0463] E. Gene-edited TILs In some embodiments, the method disclosed herein comprises gene editing at least a portion of TIL, for example, after the first amplification step, after enrichment step, after collection step, or after the second amplification step.In some embodiments, the method disclosed herein comprises gene editing the second group of TIL after the first amplification step.In some embodiments, the method disclosed herein comprises gene editing the third group of TIL after enrichment step, and enrichment step comprises: (a) co-culturing the TIL from the first amplification step with autologous tumor digest or tumor lysate; (b) co-culturing the TIL from the first amplification step with mature dendritic cells (which have previously been cultured with autologous tumor antigens in the form of tumor digest / tumor lysate or isolated peptide); or (c) co-culturing the TIL from the first amplification step with autologous tumor organoid or organoid, so that tumor-reactive TIL population is enriched. In some embodiments, the methods disclosed herein include gene editing a plurality of tumor-reactive TILs after the plurality of tumor-reactive TILs are separated from non-tumor-reactive TILs. In some embodiments, the methods disclosed herein include gene editing a fourth population of TILs after a second amplification step.

[0464] As used herein, "gene-editing," "gene editing," and "genome editing" refer to a type of genetic modification in which DNA is permanently modified in a cell's genome (e.g., DNA is inserted, deleted, modified, or replaced within a cell's genome). In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits (or reduces (sometimes referred to as gene knockdown)) expression of a DNA sequence. According to embodiments of the present invention, gene editing techniques are used to enhance the efficacy of therapeutic populations of TILs. Exemplary gene editing processes / methods of the present invention, and gene-edited TIL products, can be found in International Patent Application No. PCT / US22 / 14425, U.S. Provisional Application Nos. 63 / 304,498, and 63 / 242,373 (all of which are incorporated herein by reference in their entirety for all relevant purposes).

[0465] In some embodiments of the invention relating to methods of expanding a TIL population, the method includes one or more steps of introducing into at least a portion of the TILs a nucleic acid (e.g., mRNA) for transient expression of an immunomodulatory protein (e.g., an immunomodulatory fusion protein comprising an immunomodulatory protein fused to a membrane anchor) to produce modified TILs with (i) reduced dependency on cytokines when expanded in culture and / or (ii) enhanced therapeutic efficacy. As used herein, "transient gene-editing" and "transient gene editing" refer to the process of gene editing. "transient phenotypic alteration," "transient phenotypic alteration," "transient phenotypic alteration," "transient phenotypic alteration," "transient cellular alteration," "transient cellular modification," "transient cellular alteration," "transient cellular modification," "transient expression," "transient expression alteration," "transient protein expression alteration," "transient modification," "transient phenotypic alteration," "non-permanent phenotypic alteration," "transiently modified," "transiently modified," "non-permanently modified," "transiently altered," "transiently altered," grammatical variations of any of the foregoing, and any expressions of similar import refer to a type of cellular modification or phenotypic change in which a nucleic acid (e.g., mRNA) is introduced into a cell (e.g., by transferring a nucleic acid into a cell by electroporation, calcium phosphate transfection, viral transduction, etc.) and expressed in the cell (e.g., expression of an immunomodulatory protein, such as an immunomodulatory fusion protein comprising an immunomodulatory protein fused to a membrane anchor) to achieve a transient or non-permanent phenotypic change in the cell (e.g., transient display of a membrane-anchored immunomodulatory fusion protein on the cell surface). According to embodiments of the present invention, transient phenotype alteration techniques are used to reduce the dependency on cytokines for expansion of TILs in culture and / or to enhance the efficacy of therapeutic populations of TILs.

[0466] In some embodiments, a microfluidic platform is used for intracellular delivery of a nucleic acid encoding an immunomodulatory fusion protein provided herein. In some embodiments, the microfluidic platform is an SQZ vector-free microfluidic platform. The SQZ platform is capable of delivering nucleic acids and proteins to various primary human cells, including T cells (Sharei et al. PNAS 2013, Sharei et al. PLOS ONE 2015, and Greisbeck et al. J. Immunology vol. 195, 2015). In the SQZ platform, the cell membrane of the cell to be modified (e.g., TIL) is temporarily disrupted by microfluidic constriction, thereby enabling delivery of the nucleic acid encoding the immunomodulatory fusion protein into the cell. Such methods can be used according to the invention to deliver nucleic acids encoding the immunomodulatory fusion protein of interest to a population of TILs, as described in International Patent Application Publication Nos. 2013 / 059343A1, 2017 / 008063A1, or 2017 / 123663A1, or U.S. Patent Application Publication Nos. 2014 / 0287509A1, 2018 / 0201889A1, or 2018 / 0245089A1 (incorporated herein by reference in their entireties). In some embodiments, the delivered nucleic acid allows for transient protein expression of the immunomodulatory fusion protein in the modified TILs. In some embodiments, the SQZ platform is used to stably integrate the delivered nucleic acid encoding the immunomodulatory fusion protein into the TIL cell genome. Additional exemplary disclosure of the SQZ platform and its uses can be found in International Patent Application Publication No. WO / 2019 / 136456, which is incorporated herein by reference in its entirety for all purposes.

[0467] As discussed above, embodiments of the present invention provide genetically modified tumor-infiltrating lymphocytes (TILs) via gene editing (e.g., expression of an immunomodulatory fusion protein on the cell surface) to promote a therapeutic effect. Embodiments of the present invention encompass genetic editing via the insertion of nucleotides (RNA or DNA) into a population of TILs to both enhance expression of one or more proteins and inhibit expression of one or more proteins, as well as combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into a therapeutic population, which methods involve genetically editing the TILs. There are several gene editing techniques that can be used to genetically modify a population of TILs, which are suitable for use in accordance with the present invention.

[0468] In some embodiments, the method for genetically modifying a population of TILs comprises the step of stable integration of a gene for the production of one or more proteins. In some embodiments, the method for genetically modifying a population of TILs comprises the step of retroviral transduction. In some embodiments, the method for genetically modifying a population of TILs 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. Pat. No. 6,627,442 (the disclosures of each of which are incorporated herein by reference). In some embodiments, the method for genetically modifying a population of TILs comprises a 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 some embodiments, the method for genetically modifying a population of TILs comprises a step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein (e.g., a transposase provided as mRNA (e.g., an mRNA including a cap and polyA tail)) such that long-term expression of the transposase does not occur 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) and engineered enzymes with increased enzymatic activity, are described, for example, in Hackett, et al., Mol. Therapy 2010, 18,674-83 and U.S. Pat. No. 6,489,458 (the disclosures of each of which are incorporated herein by reference).

[0469] In some embodiments, the method for genetically modifying a population of TILs comprises 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 population of TILs comprises the step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014 / 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 (these disclosures 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 some embodiments, the electroporation method includes treating a TIL with a pulsed electric field to modify, manipulate, or cause defined and controlled, permanent, or temporary changes in the TIL, and includes applying to the TIL a sequence of at least three single, operator-controlled, independently programmed DC electric pulses having an electric field strength of 100 V / cm or greater, wherein the sequence of the at least three DC electric pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method includes treating the TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled, permanent, or temporary changes in the TIL, the pulsed electroporation method including applying to the TIL a sequence of at least three single, operator-controlled, independently programmed DC electrical pulses having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses differ from each other in pulse amplitude.In some embodiments, the electroporation method includes treating the TIL with a pulsed electric field to modify, manipulate, or cause defined and controlled, permanent, or temporary changes in the TIL, the pulsed electroporation method including applying to the TIL a sequence of at least three single, operator-controlled, independently programmed DC electrical pulses having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method includes treating a TIL with a pulsed electric field to alter, manipulate, or cause defined and controlled, permanent, or temporary changes in the TIL, the pulsed electroporation method including applying to the TIL a sequence of at least three single, operator-controlled, independently programmed DC electrical pulses having a field strength of 100 V / cm or greater, wherein a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses.In some embodiments, the electroporation method comprises treating TILs with a pulsed electric field to induce pore formation in the TILs, the pulsed electroporation method comprising applying to the TILs a sequence of at least three DC electric pulses having an electric field strength of 100 V / cm or more, wherein the sequence of the at least three DC electric pulses has one, two, or three of the following characteristics so that the induced pores are sustained for a relatively long period of time and the viability of the TILs is maintained: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the method for genetically modifying a population of TILs 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 some embodiments, the method for genetically modifying a population of TILs includes a liposome 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 dioleoylphophotidylethanolamine (DOPE) in filtered water) 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 some embodiments, the method for genetically modifying a population of TILs comprises a transfection step using the method 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).

[0470] According to some embodiments, the gene editing process may involve the use of a programmable nuclease that mediates the generation of double-strand or single-strand breaks in one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci (i.e., the nuclease relies on the recognition of specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence). The double-strand break in DNA subsequently recruits endogenous repair mechanisms to the break site to mediate genome editing by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Thus, repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, suppress, or promote) the targeted gene product.

[0471] 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) are targeted to specific DNA sequences by 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.

[0472] Non-limiting examples of gene editing methods that can be used according to the TIL expansion methods of the present invention include CRISPR, TALE, and ZFN methods, embodiments of which are described in more detail below. According to some embodiments, the method of amplifying TILs into a therapeutic population can be performed according to any embodiment of the methods described herein (e.g., Process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, and the method further includes gene editing at least a portion of the TILs using one or more CRISPR methods (TALE or ZFN methods) to generate TILs that can provide an enhanced therapeutic effect. According to some embodiments, the gene-edited TILs can be evaluated for improved therapeutic effect by comparing them in vitro to unmodified TILs (e.g., by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs).

[0473] In some embodiments of the present invention, electroporation is used for the delivery of 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 ECM 830 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 forms a closed, sterile system with the rest of the TIL amplification method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, which together with the rest of the TIL amplification method form a closed, sterile system.

[0474] In some embodiments, a microfluidic platform is used for delivery of the gene editing system. In some embodiments, the microfluidic platform is an SQZ vector-free microfluidic platform. a.CRISPR method

[0475] The method of amplifying TILs into a therapeutic population can be carried out according to any embodiment of the methods described herein (e.g., Process 2A), or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, and further comprises gene editing at least a portion of the TILs with CRISPR methods (e.g., CRISPR / Cas9 or CRISPR / Cpfl). According to certain embodiments, the use of CRISPR methods during the TIL expansion process causes expression of at least one immunomodulatory composition on the cell surface of at least a portion of the therapeutic population of TILs, and optionally causes one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic population of TILs. Alternatively, use of CRISPR methods during the TIL expansion process causes expression of at least one immunomodulatory composition on the cell surface of at least a portion of the therapeutic population of TILs, optionally causing one or more immune checkpoint genes to be upregulated in at least a portion of the therapeutic population of TILs. In some embodiments, the at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane-anchored immunomodulatory fusion protein described herein). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., CD40L or an agonistic CD40 binding domain). In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist. In some embodiments, the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.

[0476] CRISPR stands for "clustered regularly interspaced short palindromic repeats." Methods using CRISPR systems for gene editing are also referred to herein as CRISPR methods. CRISPR systems can be divided into two major classes (Class 1 and Class 2), which are further classified into different types and subtypes. The classification of CRISPR systems is based on the effector Cas protein capable of cleaving specific nucleic acids. In Class 1 CRISPR systems, the effector module consists of a multiprotein complex, while Class 2 systems use only one effector protein. Class 1 CRISPRs include Type I, Type III, and Type IV, while Class 2 CRISPRs include Type II, Type V, and Type VI. While any of these types of CRISPR systems can be used in accordance with the present invention, there are three types of CRISPR systems incorporating RNA and Cas proteins that are preferred for use in accordance with the present invention: Type I (exemplified by Cas3), Type II (exemplified by Cas9), and Type III (exemplified by Cas10). Type II CRISPR is one of the best-characterized systems.

[0477] CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea (a domain of unicellular microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to thwart viral attacks and other foreign agents by chopping up and destroying the invader's DNA. CRISPRs are specialized regions of DNA with two distinct features: nucleotide repeats and the presence of spacers. Repeated sequences of nucleotides are distributed throughout the CRISPR region, and short segments of foreign DNA (spacers) are interspersed among the repeats. In Type II CRISPR / Cas systems, the spacers are integrated into the CRISPR genomic locus and transcribed and processed into short CRISPR RNAs (crRNAs). These crRNAs anneal to trans-activating crRNAs (tracrRNAs) and direct the sequence-specific cleavage and silencing of pathogenic DNA by Cas proteins. Target recognition by the Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA-binding region. The CRISPR / Cas system can thereby be retargeted to cleave virtually any DNA sequence by redesigning the crRNA. Thus, according to certain embodiments, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA recognition. The crRNA and tracrRNA in the native system can be simplified to a single guide RNA (sgRNA) of approximately 100 nucleotides for use in genetic engineering. The sgRNA is a synthetic RNA that contains a scaffold sequence required for Cas binding and a user-defined, approximately 17-20 nucleotide spacer that defines the genomic target to be modified. Thus, users can alter the genomic target of the Cas protein by altering the target sequence present in the sgRNA. The CRISPR / Cas system can be directly delivered to human cells by co-delivery of plasmids expressing the Cas9 endonuclease and RNA components (e.g., sgRNA).Different variants of Cas proteins can be used to reduce targeting restriction (e.g., orthologues of Cas9 such as Cpf1).

[0478] According to some embodiments, the engineered, programmable, non-naturally occurring Type II CRISPR-Cas system includes a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a TIL, where the DNA molecule encodes at least one immune checkpoint molecule, the TIL expresses the molecule, and the Cas9 protein cleaves the DNA molecule, thereby altering expression of the at least one immune checkpoint molecule, where the Cas9 protein and the guide RNA do not naturally occur together. According to some embodiments, the expression of two or more immune checkpoint molecules is altered. According to some embodiments, the guide RNA(s) include a guide sequence fused to a tracr sequence. For example, the guide RNA may include a crRNA-tracrRNA or an sgRNA. According to aspects of the present invention, the terms "guide RNA," "single guide RNA," and "synthetic guide RNA" are used interchangeably and may refer to a polynucleotide sequence that includes a guide sequence of approximately 17-20 bp within the guide RNA that specifies a target site.

[0479] Cas9 variants with improved on-target specificity compared to Cas9 can also be used in accordance with embodiments of the present invention. Such variants can be referred to as high-fidelity Cas9s. According to some embodiments, a dual-nickase approach can be utilized, in which two nickases targeting opposite DNA strands generate DSBs within the target DNA (often referred to as a double-nick CRISPR system or dual-nickase CRISPR system). For example, this approach can involve a mutation in one of the two Cas9 nuclease domains, converting Cas9 from a nuclease to a nickase. Non-limiting examples of high-fidelity Cas9s include eSpCas9, SpCas9HF1, and HypaCas9. Such variants can reduce or eliminate unwanted changes at non-target DNA sites. See, e.g., Slaymaker IM, et al. Science. 2015 Dec 1, Kleinstiver BP, et al. Nature. 2016 Jan 6, and Ran et al., Nat Protoc. 2013 Nov;8(11):2281-2308, the disclosures of which are incorporated herein by reference.

[0480] Additionally, according to certain embodiments, Cas9 scaffolds can be used that improve Cas9 gene delivery into cells and improve on-target specificity, such as those disclosed in U.S. Patent Application Publication No. 2016 / 0102324, which is incorporated herein by reference. For example, the Cas9 scaffold can include a RuvC motif defined by (D-[I / L]-GXXSXGWA) and / or an HNH motif defined by (YXXDHXXPXSXXXDXS), where X represents any one of the 20 naturally occurring amino acids and [I / L] represents isoleucine or leucine. The HNH domain is responsible for nicking one strand of the target dsDNA, and the RuvC domain is involved in cleaving the other strand of the dsDNA. Thus, each of these domains nicks the strand of the target DNA within the protospacer immediately adjacent to the PAM, resulting in blunt DNA cleavage. These motifs can be combined with each other to generate more compact and / or more specific Cas9 scaffolds. Furthermore, the motifs can be used to generate split-Cas9 proteins split into two separate RuvC and HNH domains (i.e., reduced or truncated forms of the Cas9 protein or Cas9 variants containing either the RuvC or HNH domains), which can process target DNA together or separately.

[0481] According to certain embodiments, the CRISPR method involves silencing or reducing expression of one or more immune checkpoint genes in TILs by introducing a Cas9 nuclease and a guide RNA (e.g., crRNA-tracrRNA or sgRNA) containing a sequence of approximately 17-20 nucleotides specific to the target DNA sequence of the immune checkpoint gene(s). The guide RNA can be delivered as RNA or by transformation of a plasmid with the guide RNA coding sequence under a promoter. The CRISPR / Cas enzyme introduces a double-strand break (DSB) at a specific location based on the sgRNA-defined target sequence. DSBs can be repaired in cells by non-homologous end joining (NHEJ), a mechanism that frequently causes insertions or deletions (indels) in DNA. Indels often lead to frameshifts, generating loss-of-function alleles, for example, by causing a premature stop codon within the open reading frame (ORF) of the targeted gene. According to certain embodiments, the result is a loss-of-function mutation in the targeted immune checkpoint gene.

[0482] Alternatively, DSBs induced by CRISPR / Cas enzymes 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 using homology-directed repair (HDR), specific nucleotide changes ranging from single nucleotide changes to large insertions can be generated. According to some embodiments, HDR is used to gene-edit immune checkpoint genes by delivering a DNA repair template containing the desired sequence into TILs together with sgRNA(s) and Cas9 or Cas9 nickase. The repair template preferably contains additional homologous sequences (often referred to as left and right homologous arms) immediately upstream and downstream of the desired edited and target gene.

[0483] According to certain embodiments, an enzymatically inactive version of Cas9 (deadCas9 or dCas9) can be targeted to a transcription start site to suppress transcription by blocking initiation. Thus, targeted immune checkpoint genes can be suppressed without the use of DSBs. The dCas9 molecule retains the ability to bind to target DNA based on the sgRNA target sequence. According to some embodiments of the present invention, CRISPR methods include silencing or reducing the expression of one or more immune checkpoint genes by inhibiting or preventing transcription of the targeted gene(s). For example, CRISPR methods can include fusing a transcription repressor domain, such as a Kruppel-associated box (KRAB) domain, to an enzymatically inactive version of Cas9, thereby forming dCas9-KRAB, which targets, for example, the transcription start site of an immune checkpoint gene, leading to the inhibition or prevention of gene transcription. Preferably, the repressor domain is targeted to a downstream window (e.g., about 500 bp downstream) from the transcription start site. This approach, which may be referred to as CRISPR interference (CRISPRi), leads to robust gene knockdown via reduced transcription of targeted RNA.

[0484] According to certain embodiments, an enzymatically inactive version of Cas9 (deadCas9 or dCas9) can be targeted to a transcription start site to activate transcription. This approach can be referred to as CRISPR activation (CRISPRa). According to some embodiments, the CRISPR method involves increasing expression of one or more immune checkpoint genes by activating transcription of the targeted genes. According to such embodiments, the targeted immune checkpoint genes can be activated without the use of DSBs. The CRISPR method can involve targeting a transcription activation domain to the transcription start site, for example, by fusing a transcription activator (such as VP64) to dCas9, thereby forming dCas9-VP64, which targets the transcription start site of an immune checkpoint gene, thereby activating transcription of the gene. Preferably, the activator domain is targeted to a window upstream (e.g., approximately 50-400 bp downstream) from the transcription start site.

[0485] Additional embodiments of the present invention may utilize activation strategies that have been developed for potent activation of target genes in mammalian cells. Non-limiting examples include co-expression of epitope-tagged dCas9 and antibody activator effector proteins (e.g., the SunTag system), dCas9 fused to multiple different activation domains in series (e.g., dCas9-VPR), or co-expression of dCas9-VP64 with a modified scaffold gRNA and additional RNA-binding helper activators (e.g., SAM activators).

[0486] According to another embodiment, a CRISPR-mediated genome editing method called CRISPR-assisted rational protein engineering (CARPE), as disclosed in U.S. Patent No. 9,982,278 (which is incorporated herein by reference), can be used in accordance with embodiments of the present invention. CARPE involves the generation of a "donor" library and a "destination" library that directly incorporate mutations directed from single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) editing cassettes into the genome. Construction of the donor library involves co-transforming rationally designed editing oligonucleotides into cells with guide RNAs (gRNAs) that hybridize to the target DNA sequence. The editing oligonucleotides are designed to couple the deletion or mutation of the PAM with mutations of one or more desired codons in adjacent genes. This allows the entire donor library to be generated in a single transformation. The donor library is retrieved by amplification of the recombinant chromosomes (e.g., by PCR reaction) using a synthetic feature from the editing oligonucleotide (i.e., a second PAM deletion or mutation simultaneously incorporated at the 3' end of the gene). This covalently couples the codon-targeted mutation directed to the PAM deletion. The donor library is then cotransformed into cells with the destination gRNA vector to generate a cell population expressing the rationally designed protein library.

[0487] According to another embodiment, a method for traceable precise genome editing using a CRISPR-mediated system, referred to as genome engineering by traceable CRISPR enrichment recombineering (GEn-TraCER), as disclosed in U.S. Patent No. 9,982,278 (which is incorporated herein by reference), can be used in accordance with embodiments of the present invention. The GEN-TraCER method and vector combines an editing cassette with a gene encoding a gRNA on a single vector. The cassette contains the desired mutation and a PAM mutation. A vector, which may also encode Cas9, is introduced into a cell or cell population. This activates expression of the CRISPR system in the cell or cell population, allowing the gRNA to recruit Cas9 to the target region, where dsDNA cleavage occurs, allowing for integration of the PAM mutation.

[0488] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing TILs via CRISPR methods 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, TNFRSF10C, TNFRSF10D, TNFRSF10E, TNFRSF10F, TNFRSF10G, TNFRSF10H, TNFRSF10I ... FRSF10A, 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.

[0489] Non-limiting examples of genes that can be enhanced by permanently gene editing TILs via CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

[0490] The system, method and composition example for modifying the expression of target gene sequence by CRISPR method and can be used according to the present invention embodiment are described in United States Patent No. 8,697,359; United States Patent No. 8,993,233; United States Patent No. 8,795,965; United States Patent No. 8,771,945; United States Patent No. 8,889,356; United States Patent No. 8,865,406; United States Patent No. 8,999,641; United States Patent No. 8,945,839; United States Patent No. 8,932,814; United States Patent No. 8,871,445; United States Patent No. 8,906,616; and United States Patent No. 8,895,308 (which are incorporated herein by reference).The resources for implementing CRISPR method (such as the plasmid for expressing CRISPR / Cas9 and CRISPR / Cpf1) are commercially available from companies such as GenScript.

[0491] In some embodiments, the genetic modification of the population of TILs described herein can be accomplished using the CRISPR / Cpf1 system as described in U.S. Patent No. 9,790,490 (the disclosure of which is incorporated herein by reference). The CRISPR / Cpf1 system is functionally different from the CRISPR-Cas9 system in that the Cpf1-associated CRISPR array is processed into mature crRNA without the need for additional tracrRNA. The crRNA used in the CRISPR / Cpf1 system has a spacer or guide sequence and direct repeat sequences. The Cpf1p-crRNA complex formed using this method is sufficient by itself to cleave the target DNA.

[0492] b.TALE method The method for expanding TILs into a therapeutic population can be carried out according to any embodiment of the methods described herein (e.g., Process 2A), or as described in WO2018081473, WO2018129332, or WO2018182817, and further comprises gene editing at least a portion of the TILs using the TALE method. According to certain embodiments, use of the TALE method during the TIL expansion process causes expression of at least one immunomodulatory composition on the cell surface in at least a portion of the therapeutic population of TILs, and optionally causes expression of one or more immune checkpoint genes to be silenced or reduced. Alternatively, use of the TALE method during the TIL expansion process causes expression of at least one immunomodulatory composition on the cell surface in at least a portion of the therapeutic population of TILs, and optionally causes expression of one or more immune checkpoint genes to be promoted.

[0493] TALE stands for "transcription activator-like effector" protein, which includes TALENs ("transcription activator-like effector nucleases"). Methods using the TALE system for gene editing may also be referred to herein as TALE methods. TALEs are naturally occurring proteins from the plant pathogenic bacterium Xanthomonas genus, and 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 repeats are linked together to recognize contiguous DNA sequences. Specific RVDs in the DNA-binding domain recognize bases in the target locus and provide structural features for assembling a predictable DNA-binding domain. The DNA-binding domain of a TALE is fused to the catalytic domain of a type IIS FokI endonuclease to create a targetable TALE nuclease. To induce site-specific mutations, two individual TALEN arms (separated by a 14–20 base pair spacer region) dimerize with FokI monomers in close proximity to produce a targeted double-stranded break.

[0494] Multiple large-scale systematic studies utilizing various assembly methods have shown that TALE repeats can be combined to recognize virtually any user-defined sequence. Strategies that enable rapid assembly of custom TALE arrays include Golden Gate molecular cloning, high-throughput solid-phase assembly, and ligation-independent cloning techniques. Custom-designed TALE arrays are also commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Additionally, web-based tools (such as TAL Effector-Nucleotide Target 2.0) are available that allow the design of custom TAL effector repeat arrays for desired targets and also provide predicted TAL effector binding sites. See Doyle, et al., Nucleic Acids Research, 2012, Vol. 40, W117-W122. Examples of TALE and TALEN methods suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 2011 / 0201118A1; 2013 / 0117869A1; 2013 / 0315884A1; 2015 / 0203871A1, and 2016 / 0120906A1, the disclosures of which are incorporated herein by reference.

[0495] According to some embodiments of the present invention, the TALE method comprises silencing or reducing expression of one or more immune checkpoint genes by inhibiting or preventing transcription of the targeted gene(s). For example, the TALE method may involve utilizing KRAB-TALE, which comprises fusing a transcriptional Kruppel-associated box (KRAB) domain to a DNA-binding domain that targets the transcription start site of the gene, leading to the inhibition or prevention of transcription of the gene.

[0496] According to other embodiments, the TALE method involves silencing or reducing the expression of one or more immune checkpoint genes by introducing mutations into the targeted gene(s). For example, the TALE method may involve fusing a nuclease effector domain (such as Fokl) to a TALE DNA-binding domain, resulting in a TALEN. Fokl is active as a dimer, and therefore the method involves constructing a pair of TALENs to position the FOKL nuclease domain at adjacent genomic target sites, where they introduce a DNA double-strand break. Following the correct location and dimerization of Fokl, the double-strand break can be completed. Once the double-strand break is introduced, DNA repair can be achieved via two different mechanisms: high-fidelity homologous recombination pairing (HRR) (also known as homology-directed repair or HDR) or error-prone non-homologous end joining (NHEJ). Repair of the double-strand break via NHEJ preferably results in a deletion, insertion, or replacement of the DNA target site. That is, NHEJ typically introduces small insertions and deletions at the site of the break, often resulting in a frameshift that knocks out gene function. According to certain embodiments, a TALEN pair is targeted to the 5'-most exon of a gene to promote a premature frameshift mutation or a premature stop codon. The genetic mutation(s) introduced by the TALEN are preferably permanent. Thus, according to some embodiments, a method includes using dimerized TALENs to induce a site-specific double-strand break that is repaired via error-prone NHEJ, thereby silencing or reducing the expression of an immune checkpoint gene by introducing one or more mutations in the targeted immune checkpoint gene.

[0497] According to additional embodiments, TALENs are used to introduce genetic alterations (such as non-random point mutations, targeted deletions, or addition of DNA fragments) via HRR. The introduction of DNA double-strand breaks allows for gene editing via homologous recombination in the presence of suitable donor DNA. According to some embodiments, the method includes co-delivering a dimerized TALEN and a donor plasmid with locus-specific homology arms to induce site-specific double-strand breaks and integrate one or more transgenes into the DNA.

[0498] According to another embodiment, TALENs, hybrid proteins derived from FokI and AvrXa7, as disclosed in U.S. Patent Publication No. 2011 / 0201118, can be used in accordance with embodiments of the present invention. This TALEN retains the recognition specificity of AvrXa7 for target nucleotides and the double-stranded DNA cleavage activity of FokI. Using the same method, other TALENs with different recognition specificities can be prepared. For example, compact TALENs can be generated by engineering a core TALE scaffold with a different set of RVDs to alter DNA binding specificity and target a specific single dsDNA target sequence. See U.S. Patent Publication No. 2013 / 0117869. A selection of catalytic domains can be attached to the scaffold to achieve DNA processing, which can be engineered to ensure that the catalytic domain, when fused to the core TALE scaffold, is capable of processing DNA in the vicinity of a single dsDNA target sequence. Peptide linkers can also be engineered to fuse catalytic domains to scaffolds to generate compact TALENs consisting of a single polypeptide chain that do not require dimerization to target a specific single dsDNA sequence. The core TALE scaffold can also be modified by fusing a catalytic domain (which can be a TAL monomer) to its N-terminus, allowing this catalytic domain to potentially interact with another catalytic domain fused to another TAL monomer, thereby generating a catalytic entity that can process DNA adjacent to the target sequence. See US Patent Publication No. 2015 / 0203871. This architecture allows only one DNA strand to be targeted, which is not an option for classical TALEN architecture.

[0499] According to some embodiments of the present invention, conventional RVDs can be used to generate TALENs capable of significantly reducing gene expression. In some embodiments, four RVDs (NI, HD, NN, and NG) are used to target adenine, cytosine, guanine, and thymine, respectively. These conventional RVDs can be used to generate TALENs that target, for example, the PD-1 gene. Examples of TALENs that use conventional RVDs include the T3v1 TALEN and T1 TALEN disclosed in Gautron et al., Molecular Therapy: Nucleic Acids Dec. 2017, Vol. 9: 312-321 (Gautron), which is incorporated herein by reference. The T3v1 TALEN and T1 TALEN target the second exon of the PDCD1 gene locus, where the PD-L1 binding site is located, and can significantly reduce PD-1 production. In some embodiments, the T1 TALEN does so by using target sequence number 256, and the T3v1 TALEN does so by using target sequence number 257.

[0500] According to another embodiment, TALENs are modified with non-conventional RVDs to improve their activity and specificity for target genes, as disclosed in Gautron. Naturally occurring RVDs cover only a small proportion of the possible diversity repertoire for hypervariable amino acid positions. Non-conventional RVDs provide alternatives to natural RVDs and have novel, unique targeting specificity features that can be used to exclude TALEN targeting of off-site targets (sequences in the genome that contain a small number of mismatches compared to the targeted sequence). Non-conventional RVDs can be identified by generating and screening a collection of TALENs containing alternative combinations of amino acids at two hypervariable amino acid positions at defined positions in an array, as disclosed in Juillerat, et al., Scientific Reports 5, Article Number 8150 (2015) (which is incorporated herein by reference). Non-conventional RVDs that discriminate between nucleotides present at the mismatched positions can then be selected, which can prevent TALEN activity at off-site sequences while still allowing proper processing of the target position. Then, the selected non-traditional RVD can be used to replace the conventional RVD in TALEN.Examples of TALENs in which conventional RVDs are replaced by non-traditional RVDs include the T3v2 PD-1 TALEN and T3v3 PD-1 TALEN produced by Gautron.These TALENs have increased specificity when compared with the TALENs that use conventional RVDs.

[0501] According to additional embodiments, TALENs can be used to introduce genetic alterations that silence or reduce the expression of two genes. For example, two separate TALENs can be generated to target two different genes and then used together. The molecular events generated by the two TALENs at their respective loci and potential off-target sites can be characterized by high-throughput DNA sequencing. This allows for analysis of off-target sites and site identification resulting from the use of both TALENs. Based on this information, appropriate conventional and non-conventional RVDs can be selected to engineer TALENs with increased specificity and activity even when used together. For example, Gautron discloses the combined use of a T3v4 PD-1 TALEN and a TRAC TALEN to produce double knockout CAR T cells that maintain potent in vitro anti-tumor function.

[0502] In some embodiments, the Gautron method or other methods described herein can be used to genetically edit TILs, which can then be amplified by any of the procedures described herein.

[0503] According to other embodiments, TALENs can be specifically designed, allowing for a higher rate of DSB events in target cells that can target a specific selection of genes. See U.S. Patent Publication No. 2013 / 0315884. The use of such low-frequency-cutting endonucleases increases the chance of obtaining double inactivation of target genes in transfected cells, enabling the production of engineered cells (such as T cells). Furthermore, additional catalytic domains can be introduced by TALENs to increase mutagenesis and facilitate target gene inactivation. The TALENs described in U.S. Patent Publication No. 2013 / 0315884 have been successfully used to engineer T cells to make them suitable for immunotherapy. TALENs can also be used to inactivate various immune checkpoint genes in T cells, including inactivating at least two genes in a single T cell. See U.S. Patent Publication No. 2016 / 0120906. Additionally, TALENs can be used to inactivate genes encoding targets for immunosuppressants and T cell receptors, as disclosed in U.S. Patent Publication No. 2018 / 0021379 (which is incorporated herein by reference). Furthermore, TALENs can be used to inhibit the expression of beta2-microglobulin (B2M) and / or class II major histocompatibility complex transactivator (CIITA), as disclosed in U.S. Patent Publication No. 2019 / 0010514 (which is incorporated herein by reference).

[0504] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing TILs via the TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNF These include RSF10A, 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.

[0505] c. Zinc finger method The method of expanding TILs into a therapeutic population can be carried out according to any embodiment of the methods described herein (e.g., Process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, and further comprises gene editing at least a portion of the TILs by zinc finger or zinc finger nuclease methods. According to certain embodiments, use of zinc finger methods during the TIL expansion process causes expression of at least one immunomodulatory composition on the cell surface in at least a portion of the therapeutic population of TILs, and optionally causes expression of one or more immune checkpoint genes to be silenced or reduced. Alternatively, use of zinc finger methods during the TIL expansion process causes expression of at least one immunomodulatory composition on the cell surface in at least a portion of the therapeutic population of TILs, and optionally causes expression of one or more immune checkpoint genes to be enhanced.

[0506] Each zinc finger contains approximately 30 amino acids in a conserved ββα configuration. Multiple amino acids on the surface of the α-helix typically contact 3 bp in the major groove of DNA with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA-binding domain containing the zinc finger, which includes eukaryotic transcription factors. The second domain is a nuclease domain responsible for catalytic cleavage of DNA, which includes the FokI restriction enzyme.

[0507] The DNA-binding domain of an individual ZFN typically contains three to six distinct zinc finger repeats, each capable of recognizing 9 to 18 base pairs. If the zinc finger domains are specific for their intended target site, then even a single pair of three-finger ZFNs, recognizing a total of 18 base pairs, could theoretically target a single locus in a mammalian genome. One method for generating new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The most common modular assembly process involves combining three separate zinc fingers, each capable of recognizing a 3-base pair DNA sequence, to generate a three-finger array capable of recognizing a 9-base pair target site. Alternatively, selection-based approaches such as oligomerization pool engineering (OPEN) can be used to select new zinc finger arrays from randomized libraries that take into account context-dependent interactions between adjacent fingers. Engineered zinc fingers are commercially available, and Sangamo Biosciences (Richmond, CA, USA) has partnered with Sigma-Aldrich (St. Louis, MO, USA) to develop a proprietary platform (CompoZr®) for zinc finger construction.

[0508] Non-limiting examples of genes that can be silenced or inhibited by permanently gene editing TILs via zinc finger methods 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.

[0509] Non-limiting examples of genes that may be enhanced by permanently gene editing TILs via zinc finger methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

[0510] Examples of systems, methods, and compositions for altering expression of target gene sequences by zinc finger methods and that may be used in accordance with embodiments of the present invention are described in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, and 6,933,113. Nos. 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 479,626, which are incorporated herein by reference.

[0511] Other examples of systems, methods, and compositions for altering expression of target gene sequences by zinc finger methods and that may be used in accordance with embodiments of the present invention are described in Beane, et al., Mol. Therapy, 2015, 23 1380-1390, the disclosure of which is incorporated herein by reference.

[0512] d.Cas-CLOVER method The method of expanding TILs into a therapeutic population can be carried out according to any embodiment of the methods described herein (e.g., Process 2A), or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, and further comprises gene editing at least a portion of the TILs using the Cas-CLOVER method. According to certain embodiments, use of the Cas-CLOVER method during the TIL expansion process causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic population of TILs. Alternatively, use of the Cas-CLOVER method during the TIL expansion process causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic population of TILs.

[0513] Cas-CLOVER is a dimeric, high-fidelity site-specific nuclease (SSN) consisting of a fusion of catalytically inactive SpCas9 (dCas9) with the nuclease domain from the CloO51 type IIs restriction endonuclease from Clostridium spp. (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 based on the dimerization of the CloO51 nuclease domain, whose activity is enabled by RNA-guided recognition of two adjacent 20-nucleotide target sequences. Unlike paired nickase approaches, monomeric Cas-CLOVER does not introduce nicks or DSBs, for example, when using the Cas9-D10A mutant. Cas-CLOVER has been shown to have low off-target nuclease activity.

[0514] Exemplary Cas-CLOVER systems include those described in WO2019 / 126578, the contents of which are incorporated herein by reference in their entirety. In embodiments, the Cas-CLOVER system comprises a fusion protein that comprises, consists essentially of, or consists of a DNA localization component and an effector molecule.

[0515] DNA localization component In some embodiments, the DNA localization component is capable of binding a specific DNA sequence. In some 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.

[0516] In some embodiments, the DNA localization component comprises an oligonucleotide directed to a specific locus(s) in the genome. The oligonucleotide may be selected from DNA, RNA, DNA / RNA hybrids, and combinations thereof.

[0517] In some embodiments, the DNA localization component comprises a nucleotide-binding protein or protein complex that binds 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 some 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 some embodiments, the DNA localization component comprises an engineered protein capable of binding to the target DNA. In some embodiments, the DNA localization component comprises a protein capable of binding a DNA sequence selected from a meganuclease, a zinc finger array, a transcription activator-like (TAL) array, and a combination thereof. In some embodiments, the DNA localization component comprises a protein containing a naturally occurring DNA-binding domain. In some 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 some embodiments, the DNA localization component comprises an oligonucleotide directed to a specific locus in the genome. Exemplary oligonucleotides include, but are not limited to, DNA, RNA, a DNA / RNA hybrid, and any combination thereof. In some 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, an RNA polymerase, a DNA polymerase, and any combination thereof. In some embodiments, the protein or protein complex capable of recognizing an R-loop comprises Cas9.In some embodiments, the DNA localization component comprises a protein capable of binding a DNA sequence selected from a meganuclease, a zinc finger array, a TAL array, and any combination thereof, hi some embodiments, the DNA localization component comprises an oligonucleotide directed to a target location in the genome and a protein capable of binding to the target DNA sequence.

[0518] In some embodiments, the DNA localization component comprises, consists essentially of, or consists of at least one guide RNA (gRNA). In some 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 some embodiments, the DNA localization component comprises, consists essential...

Claims

1. (a) obtaining a tumor sample from a patient; (b) dividing the tumor sample into a first portion and a second portion; (c) performing a first expansion of a first population of tumor-reactive tumor-infiltrating lymphocytes (TILs) in the second portion of the tumor sample by culturing the second portion of the tumor sample in a first cell culture medium and IL-2 to produce a second population of TILs; and (d) contacting the second population of TILs with tumor cells or tumor cell antigens from or derived from a tumor digest obtained by digestion of the first portion of the tumor sample to produce a third population of TILs.

1. A method for enriching TILs comprising: The method, wherein the third population of TILs comprises a plurality of tumor-reactive TILs that are enriched compared to the second population of TILs.

2. 10. The method of claim 1, wherein step (d) is carried out for about 1 to about 3 days.

3. (a) obtaining a tumor sample from a patient; (b) dividing the tumor sample into a first portion and a second portion; (c) performing a first expansion of a first population of tumor-reactive tumor-infiltrating lymphocytes (TILs) in the second portion of the tumor sample by culturing the second portion of the tumor sample in a first cell culture medium and IL-2 to produce a second population of TILs; and (d) contacting the second population of TILs with tumor cells or tumor cell antigens from a tumor digest obtained by digestion of the first portion of the tumor sample or with a population of mature dendritic cells (DCs) generated from culturing a population of immature DCs with tumor cells or tumor cell antigens derived from the tumor digest to generate a third population of TILs, wherein the third population of TILs comprises a plurality of tumor-reactive TILs that are enriched compared to the second population of TILs.

4. The method of claim 3, wherein the population of immature DCs is generated by culturing a population of monocytes in the presence of GM-CSF and IL-4.

5. The method of claim 4, wherein the population of monocytes is obtained from PBMCs.

6. The method of claim 5 , wherein the PBMCs are obtained from the patient.

7. The method of any one of claims 4 to 6, wherein the population of monocytes is cultured in the presence of GM-CSF and IL-4 for about 6 days.

8. 8. The method of any one of claims 3 to 7, wherein culturing the population of immature DCs in the presence of tumor cells or tumor cell antigens from or derived from the tumor digest comprises generating a tumor lysate from the tumor digest, and culturing the immature DCs in the presence of tumor cells or tumor cell antigens from or derived from the tumor lysate.

9. The method of any one of claims 3 to 8, wherein the population of immature DCs is cultured with the tumor cells of the tumor digest at a ratio of about 3:

1.

10. 10. The method of any one of claims 3 to 9, wherein the population of immature DCs is cultured for about 24 hours in the presence of tumor cells or tumor cell antigens from or derived from the tumor digest.

11. The method of any one of claims 3 to 10, wherein the tumor digest or tumor lysate is subjected to dead cell removal before being cultured with the population of immature DCs.

12. 12. The method of any one of claims 3 to 11, wherein culturing the population of immature DCs in the presence of tumor cells or tumor cell antigens from or derived from the tumor digest is accomplished in the presence of TNFα, IL-6, and IL-1β.

13. 13. The method of claim 12, wherein the concentration of TNFα is about 2000 IU / ml.

14. The method according to claim 12 or 13, wherein the concentration of IL-6 is about 2000 IU / ml.

15. The method according to any one of claims 12 to 14, wherein the concentration of IL-1β is about 400 IU / ml.

16. The method of any one of claims 3 to 15, wherein the second population of TILs is cultured with the mature DCs.

17. 17. The method of any one of claims 3 to 16, wherein step (d) is carried out for about 1 to 3 days.

18. (a) obtaining a tumor sample from a patient; (b) dividing the tumor sample into a first portion and a second portion; (c) performing a first expansion of a first population of tumor-reactive tumor-infiltrating lymphocytes (TILs) in the second portion of the tumor sample by culturing the second portion of the tumor sample in a first cell culture medium and IL-2 to produce a second population of TILs; and (d) A method of enriching TILs, comprising contacting a second population of TILs with organoids generated from the first portion of the tumor sample to generate a third population of TILs, wherein the third population of TILs comprises a plurality of tumor-reactive TILs that are enriched compared to the second population of TILs.

19. 19. The method of claim 18, wherein generating organoids from the first portion of the tumor sample comprises digesting the first portion of the tumor sample to obtain a tumor digest, and generating organoids from the tumor digest.

20. Generating organoids (a) propelling the first portion of the tumor sample and an unpolymerized liquid matrix material through one or more channels of a microfluidic device; i. the microfluidic device controls the pressure, flow rate, or pressure and flow rate in the one or more channels and maintains a temperature of 20°C or less, such that tumor-derived cells or multiple tumor fragments in the tumor sample and the unpolymerized liquid matrix move through the one or more channels in a laminar flow; (b) combining tumor-derived cells or a plurality of tumor fragments and the unpolymerized liquid matrix material in the microfluidic device to form a plurality of droplets of the unpolymerized mixture; and (c) exposing the plurality of droplets of the unpolymerized mixture to a temperature greater than 25°C to polymerize the liquid matrix material and form the organoids; 20. The method of claim 18 or 19, comprising:

21. The method of any one of claims 1 to 20, further comprising identifying said plurality of tumor-reactive TILs in said third population of TILs.

22. 22. The method of claim 21, wherein identifying the plurality of tumor-reactive TILs comprises determining whether the TILs exhibit an activation signal that identifies the TILs as tumor-reactive.

23. 23. The method of claim 22, wherein the activation signal comprises an increase and / or decrease in cell surface expression of one or more proteins.

24. 24. The method of claim 23, wherein the one or more proteins are selected from the group consisting of CD3, CD4, CD8, PD-1, LAG3, Tim3, TIGIT, CD103, CD39, CD134, CD137, CD25, CD69, HLA-DR, CD107a, CD40L, Ki46, CD45RA, CCR7, and KLRG1.

25. 25. The method of claim 23 or 24, wherein the cell surface expression of the one or more proteins is determined by flow cytometry.

26. 26. The method of claim 25, wherein the flow cytometry is performed using a SONY FX 500, Miltenyi Tyto, or Miltenyi CliniMACS flow-activated cell sorter.

27. 23. The method of claim 22, wherein the activation signal comprises cell morphology.

28. 28. The method of claim 27, wherein the cell morphology is a flattened, rounded cell morphology.

29. 23. The method of claim 22, wherein the activation signal is the concentration of mitochondrial mass adjacent to the plasma membrane of the TIL.

30. The method of any one of claims 27 to 29, wherein the activation signal is determined by an imaging-based cell separation method.

31. The method of any one of claims 21 to 30, further comprising collecting the identified plurality of tumor-reactive TILs.

32. 32. The method of claim 31 , wherein collecting the plurality of tumor-reactive TILs comprises separating the plurality of tumor-reactive TILs from non-tumor-reactive TILs in the third population of TILs.

33. 33. The method of claim 32, wherein separating the plurality of tumor-reactive TILs comprises removing non-tumor-reactive TILs from the third population of TILs.

34. The method comprises: (e) performing a second expansion by culturing the third population of TILs or the collected plurality of tumor-reactive TILs in a second cell culture medium supplemented with additional IL-2, OKT-3, and antigen-presenting cells to generate a fourth population of TILs. The method of any one of claims 1 to 33, further comprising performing

35. 35. The method of any one of claims 1 to 34, wherein the first portion of the tumor sample comprises approximately one-third of the tumor sample.

36. 35. The method of any one of claims 1 to 34, wherein the second portion of the tumor sample comprises approximately one-half of the tumor sample.

37. 35. The method of any one of claims 1 to 34, wherein the second portion of the tumor sample comprises approximately one-third of the tumor sample.

38. 38. The method of any one of claims 1 to 17 and / or 19 to 37, wherein the tumor digest is subjected to 1, 2, 3, 4, 5, or 10 freeze-thaw cycles.

39. 39. The method of any one of claims 1 to 38, wherein the first portion of the tumor sample comprises at least 3 million cells.

40. 40. The method of any one of claims 34-39, wherein steps (a) through (e) are performed within a period of about 17 days to about 24 days, within a period of about 18 days to about 22 days, within a period of about 20 days to about 22 days, or within a period of about 22 days.

41. 41. The method of any one of claims 1 to 40, wherein the first cell culture medium further comprises a factor selected from the group consisting of IL-7, IL-15, IL-21, IL-12, leukemia inhibitory factor (LIF), beta fibroblast growth factor (bFGF), and combinations thereof.

42. 42. The method of any one of claims 1 to 41, wherein the second cell culture medium further comprises a factor selected from the group consisting of IL-7, IL-15, IL-21, IL-12, LIF, bFGF, 41BBL, OX40L, CD86, CD64, and combinations thereof.

43. 43. The method of any one of claims 1 to 42, wherein the tumor sample is selected from the group consisting of a solid tumor, a fine needle aspirate, and a mini-biopsy.

44. The method of any one of claims 22 to 43, wherein the activation signal comprises an increase in secreted interferon gamma (IFNγ).

45. 45. The method of any one of claims 1 to 44, wherein the cell culture medium is provided in a container selected from the group consisting of a G-container and a Xuri cellbag.

46. 46. ​​The method of any one of claims 1-45, further comprising gene editing the second population of TILs, the third population of TILs, or the plurality of tumor-reactive TILs.

47. A pharmaceutical composition for the treatment of cancer comprising a population of TILs generated using the method of any one of claims 1 to 46 and / or a fourth population of said TILs generated using the method of any one of claims 34 to 46.

48. 48. The composition of claim 47, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC) lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

49. 49. The composition of claim 47 or 48, further comprising a cryopreservation material.

50. 50. The composition of claim 49, wherein the cryopreservation agent comprises dimethyl sulfoxide.

51. 49. The composition of claim 47 or 48, further comprising a cryopreservation material and an isotonicity agent.

52. 52. The composition of claim 51, further comprising a cryopreservation agent comprising dimethyl sulfoxide and an isotonicity agent comprising sodium chloride, sodium gluconate, and sodium acetate.

53. 53. The composition of claim 52, further comprising a cryopreservation agent comprising dimethyl sulfoxide and dextran 40 and an isotonicity agent comprising sodium chloride, sodium gluconate, and sodium acetate.

54. 54. The composition of any one of claims 47 to 53, wherein the composition is provided in a sterile infusion bag.

55. 55. The composition of any one of claims 47 to 54, wherein the composition is harvested using a LOVO cell processing system.

56. 56. The composition of any one of claims 47-55, wherein the TILs are gene edited.

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