Genetic engineering of tumor-infiltrating lymphocyte, and use of the same in immunotherapy

Abstract

To provide genetic engineering of a tumor-infiltrating lymphocyte, and use thereof in immunotherapy.SOLUTION: Provided is an improved and / or shortened method for expanding a TIL and generating a therapeutic TIL population which includes a novel method for expanding a TIL population in a closed system which brings about improved efficiency, improved phenotype and increased metabolic health of the TIL in a shorter time period, and achieving reduced microorganism contamination and a reduced cost. The method can include genetically engineering at least a part of the TIL in order to enhance its therapeutic effect. The TIL finds use in a therapeutic treatment regimen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 926,147, filed October 25, 2019, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION

[0002] Described herein are methods for expanding tumor-infiltrating lymphocytes (TILs) and generating therapeutic TIL populations. Further disclosed herein are methods for gene editing TILs and the use of gene-edited TILs in the treatment of diseases such as cancer. [Background technology]

[0003] Background of the Invention

[0003] Treating bulky and resistant cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful therapeutic approach for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires large quantities of TILs, necessitating a robust and reliable method for commercialization. This has been challenging due to the technical, logistical, and regulatory challenges associated with cell expansion. IL-2-based TIL expansion, followed by "rapid expansion protocol" (REP), is becoming 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. REP can result in a 1,000-fold expansion of TILs in a 14-day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) as feeder cells, often from multiple donors, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. R TILs undergoing the EP procedure have led to successful adoptive cell therapy in melanoma patients after host immunosuppression. Current infusion eligibility parameters depend on the composition of the TILs (e.g., CD28, CD8, or CD4 positive) as well as the expansion fold and viability of the REP product. Current infusion eligibility parameters depend on the composition of the TILs (e.g., CD28, CD8, or CD4 positive) as well as the expansion fold and viability of the REP product.

[0004]

[0004] Current TIL manufacturing processes are limited by length, expense, sterility concerns, and other factors described herein, significantly limiting the potential for commercializing such processes, and for these and other reasons, no commercial processes are currently available. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes that are suitable for commercial-scale manufacturing and regulatory approval for use in human patients in multiple clinical centers. Furthermore, there is a strong demand for more effective TIL therapies that can increase patient response rates and robustness of response. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention

[0005] The present invention provides methods for expanding TILs and generating therapeutic TIL populations. According to exemplary embodiments, at least a portion of the therapeutic TIL population is gene-edited to enhance their therapeutic effect.

[0006] In one embodiment, the present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and wherein the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; (f) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; and (g) gene editing at least a portion of the TILs at any point during the method. Includes.

[0007]

[0007] In some embodiments, the method further comprises the step of cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process.

[0008]

[0008] In some embodiments, the cryopreservation process is performed using a 1:1 ratio of the collected TIL population to cryopreservation medium.

[0009] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are irradiated and allogeneic. In some embodiments, the PBMCs are added to the cell culture in step (d) on any of days 9 to 14. In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0010]

[0010] In some embodiments, the recovery in step (e) is carried out using a membrane-based cell processing system.

[0011] In some embodiments, the harvesting in step (e) is carried out using a LOVO cell processing system.

[0012] In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment having a length of about 27 mm. 3 It has a volume of

[0013] In some embodiments, the plurality of segments is about 1300 mm 3 ~about 1500mm 3 It contains about 30 to about 60 fragments with a total volume of .

[0014] In some embodiments, the plurality of segments is about 1350 mm 3 It contains approximately 50 fragments with a total volume of .

[0015] In some embodiments, the plurality of fragments includes about 50 fragments having a total mass of about 1 gram to about 1.5 grams.

[0016]

[0016] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell culture bag.

[0017] In some embodiments, the cell culture medium in step (d) further comprises IL-15 and / or IL-21.

[0018] In some embodiments, the IL-2 concentration is about 10,000 IU / mL to about 5,000 IU / mL.

[0019] In some embodiments, the IL-15 concentration is about 500 IU / mL to about 100 IU / mL.

[0020] In some embodiments, the IL-21 concentration is from about 20 IU / mL to about 0.5 IU / mL.

[0021]

[0021] In some embodiments, the infusion bag in step (f) is a HypoThermosol-containing infusion bag.

[0022]

[0022] In some embodiments, the cryopreservation medium comprises dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium comprises 7% to 10% dimethyl sulfoxide (DMSO).

[0023] In some embodiments, the first period of time in step (c) and the second period of time in step (e) are each independently carried out within a period of 10 days, 11 days, or 12 days.

[0024] In some embodiments, the first period of time in step (c) and the second period of time in step (e) are each carried out separately within a period of 11 days.

[0025] In some embodiments, steps (a) through (f) are carried out within a period of about 10 days to about 22 days.

[0026] In some embodiments, steps (a) through (f) are carried out within a period of about 20 days to about 22 days.

[0027] In some embodiments, steps (a) through (f) are carried out within a period of about 15 days to about 20 days.

[0028] In some embodiments, steps (a) through (f) are carried out within a period of about 10 days to about 20 days.

[0029] In some embodiments, steps (a) through (f) are carried out within a period of about 10 days to about 15 days.

[0030] In some embodiments, steps (a) through (f) are carried out in 22 days or less.

[0031] In some embodiments, steps (a) through (f) are carried out in 20 days or less.

[0032] In some embodiments, steps (a) through (f) are carried out in 15 days or less.

[0033] In some embodiments, steps (a) through (f) are carried out in 10 days or less.

[0034] In some embodiments, steps (a)-(f) and cryopreservation are carried out for 22 days or less.

[0035]

[0035] In some embodiments, the therapeutic TIL population collected in step (e) comprises sufficient TILs for a therapeutically effective dose of TILs.

[0036] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0037]

[0037] In some embodiments, steps (b) to (e) are performed in a single container, and performing steps (b) to (e) in a single container results in an increased TIL yield per resected tumor compared to performing steps (b) to (e) in multiple containers.

[0038] In some embodiments, the antigen-presenting cells are added to the TILs during the second period in step (d) without releasing the system.

[0039]

[0039] In some embodiments, the third TIL population in step (d) provides increased efficacy, increased interferon gamma production, increased polyclonality, increased mean IP-10 and / or increased mean MCP-1 when administered to a subject.

[0040]

[0040] In some embodiments, the third population of TILs in step (d) provides at least 5-fold or more interferon-γ production when administered to a subject.

[0041]

[0041] In some embodiments, the third TIL population in step (d) is a therapeutic TIL population comprising an increased subpopulation of effector T cells and / or central memory T cells relative to the second TIL population, and the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of expression of CD27+, expression of CD28+, longer telomeres, increased CD57 expression and decreased CD56 expression relative to the effector T cells and / or central memory T cells obtained from the second cell population.

[0042]

[0042] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population exhibit increased CD57 expression and decreased CD56 expression relative to effector T cells and / or central memory T cells obtained from the second cell population.

[0043] In some embodiments, the risk of microbial contamination is reduced compared to open systems. do.

[0044] In some embodiments, the TILs from step (g) are infused into the patient.

[0045] In some embodiments, the plurality of fragments comprises about 4 fragments.

[0046] In some embodiments, the cell culture medium further comprises a 4-1BB agonist and / or an OX40 agonist during the first expansion culture, the second expansion culture, or both.

[0047]

[0047] In some embodiments, gene editing is performed after the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

[0048]

[0048] In some embodiments, gene editing is performed before the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

[0049]

[0049] In some embodiments, gene editing is performed on TILs from one or more of the first population, the second population, and the third population.

[0050]

[0050] In some embodiments, gene editing is performed on TILs from the first expansion culture or TILs from the second expansion culture, or both.

[0051]

[0051] In some embodiments, gene editing is performed after the first expansion and before the second expansion.

[0052]

[0052] In some embodiments, gene editing is performed before step (c), before step (d) or before step (e).

[0053]

[0053] In some embodiments, the cell culture medium contains OKT-3 during the first expansion culture and / or the second expansion culture, and gene editing is performed before OKT-3 is introduced into the cell culture medium.

[0054]

[0054] In some embodiments, the cell culture medium contains OKT-3 during the first expansion culture and / or the second expansion culture, and gene editing is performed after OKT-3 is introduced into the cell culture medium.

[0055]

[0055] In some embodiments, the cell culture medium contains OKT-3 starting on the initiation day of the first expansion culture, and gene editing is performed after the TILs have been exposed to OKT-3.

[0056]

[0056] In some embodiments, gene editing causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic TIL population.

[0057]

[0057] In some embodiments, one or more immune checkpoint genes are PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SM Selected from the group including AD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, ANKRD11, SOCS1 and BCOR.

[0058]

[0058] In some embodiments, the one or more immune checkpoint genes are selected from the group including PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ and PKA.

[0059]

[0059] In some embodiments, the gene editing causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic TIL population, and the immune checkpoint genes are selected from the group including CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

[0060]

[0060] In some embodiments, gene editing involves the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks in the one or more immune checkpoint genes.

[0061]

[0061] In some embodiments, gene editing includes one or more methods selected from CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

[0062]

[0062] In some embodiments, gene editing includes CRISPR methods.

[0063]

[0063] In some embodiments, the CRISPR method is a CRISPR / Cas9 method.

[0064]

[0064] In some embodiments, gene editing includes the TALE method.

[0065]

[0065] In some embodiments, gene editing comprises zinc finger methods.

[0066] In another embodiment, the present invention provides a method of treating a subject having cancer, comprising administering expanded tumor infiltrating lymphocytes (TILs): (a) obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and wherein the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion culture is performed in a closed container providing a second gas-permeable surface area; The transition from step (c) to step (d) occurs without releasing the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the recovered TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally, cryopreserving the infusion bag containing the recovered TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the patient; and (i) gene editing at least a portion of the TILs at any time during method steps (a)-(f). and administering the compound comprising:

[0067]

[0067] In some embodiments, the therapeutic TIL population collected in step (e) comprises sufficient TILs to administer a therapeutically effective dose of TILs in step (h).

[0068] In some embodiments, the number of TILs sufficient to administer a therapeutically effective dose in step (h) is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0069] In some embodiments, the antigen presenting cell (APC) is a PBMC.

[0070] In some embodiments, the PBMCs are added to the cell culture in step (d) on any of days 9 to 14.

[0071]

[0071] In some embodiments, prior to administering the therapeutically effective dose of TIL cells in step (h), a non-myeloablative lymphodepletion regimen has been administered to the patient.

[0072] In some embodiments, the non-myeloablative lymphodepletion regimen is 60 mg / m 2 / day for 2 days, followed by cyclophosphamide at a dose of 25 mg / m 2 / day for 5 days.

[0073]

[0073] In some embodiments, the method further comprises treating the patient with a high-dose IL-2 regimen starting the day after administering the TIL cells to the patient in step (h).

[0074] In some embodiments, the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours to a tolerated dose.

[0075]

[0075] In some embodiments, the third TIL population in step (d) is a therapeutic TIL population comprising an increased subpopulation of effector T cells and / or central memory T cells relative to the second TIL population, and the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of expression of CD27+, expression of CD28+, longer telomeres, increased CD57 expression and decreased CD56 expression relative to the effector T cells and / or central memory T cells obtained from the second cell population.

[0076] In some embodiments, the effector T cells and / or central memory T cells in the therapeutic TIL population have increased CD57 expression and decreased CD56 expression relative to effector T cells and / or central memory T cells obtained from a second cell population. Present reality.

[0077]

[0077] 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, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer and renal cell carcinoma.

[0078] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, and NSCLC.

[0079] In some embodiments, the cancer is melanoma.

[0080] In some embodiments, the cancer is HNSCC.

[0081]

[0081] In some embodiments, the cancer is cervical cancer.

[0082] In some embodiments, the cancer is NSCLC.

[0083] In some embodiments, the cell culture medium further comprises a 4-1BB agonist and / or an OX40 agonist during the first expansion culture, the second expansion culture, or both.

[0084]

[0084] In some embodiments, gene editing is performed after the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

[0085]

[0085] In some embodiments, gene editing is performed before the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

[0086]

[0086] In some embodiments, gene editing is performed on TILs from one or more of the first population, the second population, and the third population.

[0087]

[0087] In some embodiments, gene editing is performed on TILs from the first expansion culture or TILs from the second expansion culture, or both.

[0088]

[0088] In some embodiments, gene editing is performed after the first expansion and before the second expansion.

[0089]

[0089] In some embodiments, gene editing is performed before step (c), before step (d) or before step (e).

[0090]

[0090] In some embodiments, the cell culture medium contains OKT-3 during the first expansion culture and / or the second expansion culture, and gene editing is performed before OKT-3 is introduced into the cell culture medium.

[0091]

[0091] In some embodiments, the cell culture medium contains OKT-3 during the first expansion culture and / or the second expansion culture, and gene editing is performed after OKT-3 is introduced into the cell culture medium.

[0092]

[0092] In some embodiments, the cell culture medium contains OKT-3 starting on the initiation day of the first expansion culture, and gene editing is performed after the TILs have been exposed to OKT-3.

[0093]

[0093] In some embodiments, gene editing causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic TIL population.

[0094] In some embodiments, the one or more immune checkpoint genes are PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10 , CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, ANKRD11, SOCS1 and BCOR.

[0095]

[0095] In some embodiments, the one or more immune checkpoint genes are selected from the group including PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ and PKA.

[0096]

[0096] In some embodiments, the gene editing causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic TIL population, and the immune checkpoint genes are selected from the group including CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

[0097]

[0097] In some embodiments, gene editing involves the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks in the one or more immune checkpoint genes.

[0098]

[0098] In some embodiments, gene editing includes one or more methods selected from CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

[0099]

[0099] In some embodiments, gene editing includes CRISPR methods.

[0100] In some embodiments, the CRISPR method comprises a CRISPR / Cas9 method. It is the law.

[0101]

[0101] In some embodiments, gene editing includes the TALE method.

[0102]

[0102] In some embodiments, gene editing comprises zinc finger methods.

[0103] In another embodiment, the present invention utilizes tumor infiltrating lymphocytes (TILs) as therapeutic T cells. A method for expanding an IL population is provided, the method comprising: (a) Adding processed tumor fragments from a tumor excised from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population; wherein the first expansion culture is carried out in a closed vessel providing a first gas permeable surface area, the first expansion culture is carried out for about 3 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times more numerous than the first TIL population, and the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion culture is performed in a closed container that provides a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) recovering the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without releasing the system; (e) transferring the recovered TIL population from step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; and (f) at any point during the method, gene editing at least a portion of the TILs. Includes.

[0104] In some embodiments, the therapeutic TIL population collected in step (d) , containing sufficient TILs for a therapeutically effective dose of TILs.

[0105] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0106] In some embodiments, the method comprises using a cryopreservation process to recover the The method further comprises the step of cryopreserving the infusion bag containing the isolated TIL population.

[0107] In some embodiments, the cryopreservation process involves freezing the recovered TIL population. This is done using a 1:1 ratio with the preservation medium.

[0108] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). do.

[0109] In some embodiments, the PBMCs are irradiated and allogeneic.

[0110] PBMCs were cultured on any of days 9 to 14 of the cell culture in step (c). 69. The method of claim 68, wherein the

[0111]

[0111] In some embodiments, the antigen-presenting cell is an artificial antigen-presenting cell.

[0112] In some embodiments, the harvesting in step (d) is performed using a LOVO cell processing system This is performed using

[0113] In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each The fragment is approximately 27 mm 3 It has a volume of

[0114] In some embodiments, the plurality of segments is about 1300 mm 3 ~about 1500m m 3 It contains about 30 to about 60 fragments with a total volume of .

[0115] In some embodiments, the plurality of segments is about 1350 mm 3 has a total volume of It contains approximately 50 fragments.

[0116] In some embodiments, the plurality of pieces comprises between about 1 gram and about 1.5 grams of total pieces. It contains approximately 50 fragments with different masses.

[0117] In some embodiments, the plurality of fragments comprises about 4 fragments.

[0118] In some embodiments, the second cell culture medium comprises a G container and Xuri cells. The culture medium is provided in a container selected from the group consisting of a culture bag.

[0119]

[0119] In some embodiments, the infusion bag in step (e) is a HypoThermosol-containing infusion bag.

[0120] In some embodiments, the first period and step (b) The second period in (c) shall be conducted separately within a period of 10, 11 or 12 days.

[0121] In some embodiments, the first period and step (b) The second period in (c) shall be conducted separately within a period of 11 days.

[0122] In some embodiments, steps (a) through (e) are carried out over a period of about 10 days to about 22 days. This will be carried out within the period.

[0123] In some embodiments, steps (a) through (e) are carried out over a period of about 10 days to about 20 days. This will be carried out within the period.

[0124] In some embodiments, steps (a) through (e) are carried out over a period of about 10 days to about 15 days. This will be carried out within the period.

[0125] In some embodiments, steps (a) through (e) are performed in 22 days or less. can be.

[0126] In some embodiments, steps (a)-(e) and cryopreservation are performed for 22 days. This is carried out as follows:

[0127] In some embodiments, steps (b) through (e) are carried out in a single vessel. and performing steps (b)-(e) in a single vessel results in an increased TIL yield per resected tumor compared to performing steps (b)-(e) in multiple vessels.

[0128] In some embodiments, the antigen-presenting cells are step-activated without releasing the system. During the second period in step (c), the TILs are added.

[0129] In some embodiments, the third population of TILs in step (d) is A therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells relative to a second TIL population, wherein the effector T cells and / or central memory T cells obtained in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression relative to the effector T cells and / or central memory T cells obtained from the second cell population.

[0130] In some embodiments, effectors obtained in a therapeutic TIL population The effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression relative to effector T cells and / or central memory T cells obtained from the second cell population.

[0131] In some embodiments, the risk of microbial contamination is reduced compared to open systems. can be.

[0132] In some embodiments, the TILs from step (e) are infused into the patient. do.

[0133]

[0133] In some embodiments, the closed vessel comprises a single bioreactor.

[0134]

[0134] In some embodiments, the closed container comprises G-REX-10.

[0135]

[0135] In some embodiments, the closed container comprises G-REX-100.

[0136] In some embodiments, in step (d), the antigen presenting cells (APCs) , are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

[0137] In some embodiments, the cell culture comprises 2.5×10 9 APCs vs. 10 0×10 6 The ratio of TILs is

[0138] In some embodiments, in step (c), the antigen presenting cells (APCs) , are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

[0139] In some embodiments, the cell culture comprises 2.5×10 9 APCs vs. 10 0×10 6 The ratio of TILs is

[0140] In some embodiments, the cell culture medium comprises a first expansion culture, a second expansion culture, and a The composition further comprises a 4-1BB agonist and / or an OX40 agonist, either in the form of a steroid or a combination thereof.

[0141] In some embodiments, the gene editing is performed using a 4-1BB agonist and / or This is performed after an OX40 agonist is introduced into the cell culture medium.

[0142] In some embodiments, the gene editing is performed using a 4-1BB agonist and / or This is performed before the OX40 agonist is introduced into the cell culture medium.

[0143] In some embodiments, the gene editing comprises a first population, a second population, and a third population. is performed on TILs from one or more of the populations.

[0144] In some embodiments, gene editing is performed on TILs or TILs from the first expansion culture. This may be performed on TILs from the second expansion culture, or on TILs from the second expansion culture, or both.

[0145] In some embodiments, gene editing is performed after the first expansion and after the second expansion. This is performed before incubation.

[0146] In some embodiments, the gene editing is performed before step (b), step (c), or ) or before step (d).

[0147] In some embodiments, the cell culture medium is added during the first expansion culture and / or the second expansion culture. The OKT-3 cells are then included in the expansion culture for 2 days, and gene editing is performed before the OKT-3 cells are introduced into the cell culture medium.

[0148] In some embodiments, the cell culture medium is OKT-3 is included in the expansion culture of 2, and gene editing is performed after OKT-3 is introduced into the cell culture medium.

[0149] In some embodiments, the cell culture medium is added starting on the initiation day of the first expansion culture. The gene editing is performed after the TILs are exposed to OKT-3.

[0150] In some embodiments, gene editing targets one or more immune checkpoints. The expression of the gene is caused to be silenced or reduced in at least a portion of the therapeutic TIL population.

[0151] In some embodiments, one or more immune checkpoint genes are -1, CTLA-4, LAG-3, HAVCR2(TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP 7, 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, ANKRD11, SOCS1 and BCOR.

[0152] In some embodiments, one or more immune checkpoint genes are -1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ and PKA.

[0153] In some embodiments, gene editing targets one or more immune checkpoints. The expression of the gene is caused to be enhanced in at least a portion of the therapeutic TIL population, and the immune checkpoint gene is selected from the group including CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

[0154] In some embodiments, gene editing is performed to target the one or more immune checkpoints. This involves the use of programmable nucleases to mediate the creation of double- or single-strand breaks in target genes.

[0155]

[0155] In some embodiments, gene editing is performed using CRISPR methods, TALE methods, etc. , zinc finger methods, and combinations thereof.

[0156]

[0156] In some embodiments, gene editing includes CRISPR methods.

[0157] In some embodiments, the CRISPR method is a CRISPR / Cas9 method. It is the law.

[0158]

[0158] In some embodiments, gene editing includes the TALE method.

[0159]

[0159] In some embodiments, gene editing involves zinc finger methods.

[0160]

[0160] In another embodiment, the present invention provides a method for the expansion of a mammalian animal, comprising: and providing a therapeutic TIL population (e.g., for use in treating cancer in a subject) expanded according to the method, wherein the therapeutic TIL population is permanently gene-edited.

[0161] In another embodiment, the present invention provides a method for treating a subject with cancer. Also provided is an expanded TIL population, which is obtained by a method comprising: (a) obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and wherein the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; (f) transferring the recovered TIL population from step (e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally, cryopreserving the infusion bag containing the recovered TIL population from step (f) using a cryopreservation process; and (h) at any point during the method, gene editing at least a portion of the TILs. and a third TIL population obtainable by a method comprising:

[0162] In some embodiments, the above method is further characterized by the methods and compositions described herein. The present invention further includes one or more of the features described in any one of the above.

[0163] In some embodiments, the cell culture medium comprises a first expansion culture, a second expansion culture, and a The composition further comprises a 4-1BB agonist and / or an OX40 agonist, either in the form of a steroid or a combination thereof.

[0164] In some embodiments, the gene editing is performed using a 4-1BB agonist and / or This is performed after an OX40 agonist is introduced into the cell culture medium.

[0165] In some embodiments, the gene editing is performed using a 4-1BB agonist and / or This is performed before the OX40 agonist is introduced into the cell culture medium.

[0166] In some embodiments, the gene editing comprises a first population, a second population, and a third population. is performed on TILs from one or more of the populations.

[0167] In some embodiments, gene editing is performed on TILs or TILs from the first expansion culture. This may be performed on TILs from the second expansion culture, or on TILs from the second expansion culture, or both.

[0168] In some embodiments, gene editing is performed after the first expansion and after the second expansion. This is performed before incubation.

[0169] In some embodiments, the gene editing is performed before step (c), step (d), ) or before step (e).

[0170] In some embodiments, the cell culture medium is The OKT-3 cells are then included in the expansion culture for 2 days, and gene editing is performed before the OKT-3 cells are introduced into the cell culture medium.

[0171] In some embodiments, the cell culture medium is added during the first expansion culture and / or the second expansion culture. OKT-3 is included in the expansion culture of 2, and gene editing is performed after OKT-3 is introduced into the cell culture medium.

[0172] In some embodiments, the cell culture medium is added starting on the initiation day of the first expansion culture. The gene editing is performed after the TILs are exposed to OKT-3.

[0173] In some embodiments, gene editing targets one or more immune checkpoints. The expression of the gene is caused to be silenced or reduced in at least a portion of the therapeutic TIL population.

[0174] In some embodiments, one or more immune checkpoint genes are -1, CTLA-4, LAG-3, HAVCR2(TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP 7, 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, ANKRD11, SOCS1 and BCOR.

[0175] In some embodiments, one or more immune checkpoint genes are -1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ and PKA.

[0176] In some embodiments, gene editing targets one or more immune checkpoints. and causing expression of a gene to be enhanced in at least a portion of the therapeutic TIL population, the immune checkpoint gene being CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or NOTCH ligand mD. LL1.

[0177] In some embodiments, gene editing is performed to target the one or more immune checkpoints. This involves the use of programmable nucleases to mediate the creation of double- or single-strand breaks in target genes.

[0178]

[0178] In some embodiments, gene editing is performed using CRISPR methods, TALE methods, etc. , zinc finger methods, and combinations thereof.

[0179]

[0179] In some embodiments, gene editing involves CRISPR methods.

[0180] In some embodiments, the CRISPR method comprises a CRISPR / Cas9 method. It is the law.

[0181]

[0181] In some embodiments, gene editing includes the TALE method.

[0182]

[0182] In some embodiments, gene editing involves zinc finger methods.

[0183] In another embodiment, the present invention utilizes tumor infiltrating lymphocytes (TILs) as therapeutic T cells. Also provided is a method for expanding an IL population, the method comprising: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing OKT-3 and / or 4-1BB agonist antibody, for about 2 to 5 days; (d) optionally, adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. The electroporation step includes PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3 ), and combinations thereof.

[0184]

[0184] In another embodiment, a method of treating a subject having cancer comprises: Also provided is a method for treating a tumor infiltrating lymphocyte (TIL) comprising administering the TIL to a patient having a tumor. (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing OKT-3 and / or 4-1BB agonist antibody, for about 2 to 5 days; (d) optionally, adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium; and (k) administering a therapeutically effective dose of the recovered population of TILs from the infusion bag to the patient. wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0185] In another embodiment, the present invention provides a method for treating a subject with cancer. providing a population of expanded cultured TILs, the expanded cultured TILs comprising: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) IL-2 and optionally an OKT-3 and / or 4-1BB agonist performing a first expansion culture by culturing the first TIL population in cell culture medium containing the antibody for about 2 to 5 days; (d) optionally, adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0186] In some embodiments, the method comprises administering to a subject a subject, the subject being a subject of administration of IL-2, OKT-3, and 4-1BB amyloid. The method includes performing a first expansion culture by culturing the first TIL population in cell culture medium containing an agonist antibody, wherein the OKT-3 and 4-1BB agonist antibody are optionally present in the cell culture medium starting on day 0 or day 1.

[0187] In another embodiment, the present invention provides a method for treating a subject with cancer. A cryopreservation composition is provided that includes a TIL population, a cryoprotective medium that includes DMSO, and an electrolyte solution.

[0188] In some embodiments, the cryopreserved composition contains one or more stabilizers (e.g., The antibody may further comprise one or more lymphocyte growth factors (e.g., IL-2), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-

[0189] In some embodiments, the cryoprotective medium and electrolyte solution comprising DMSO is It is present in a ratio of about 1.1:1 to about 1:1.1.

[0190]

[0190] In some embodiments, the cryopreserved composition is in a volume of about 30 mL to about 70 mL. The cryoprotective medium contains DMSO, an electrolyte solution in an amount of about 30 mL to about 70 mL, HSA in an amount of about 0.1 g to about 1.0 g, and IL-2 in an amount of about 0.001 mg to about 0.005 mg. [Brief explanation of the drawings]

[0191] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[0191] Process 2A shows a schematic diagram of an embodiment of a 22-day process for TIL fabrication. [Figure 2]

[0192] 1 shows a comparison of embodiments of the 1C and 2A processes for TIL fabrication. [Figure 3]

[0193] Shows the timeline of the 1C process. [Figure 4]

[0194] 1 shows a process for an embodiment of TIL therapy using Process 2A for TIL production, including administration and co-therapy steps for higher cell numbers. [Figure 5]

[0195] 1 shows a process for an embodiment of TIL therapy using Process 2A for TIL production, including administration and co-therapy steps, for lower cell numbers. [Figure 6]

[0196] 2A shows a detailed schematic diagram of an embodiment of the 2A process. [Figure 7a]

[0197] 1 shows the main steps of an embodiment of the 2A process, including a cryopreservation step. [Figure 7b]

[0197] The major steps of an embodiment of the 2A process, including a cryopreservation step, are shown. [Figure 7c]

[0197] The major steps of an embodiment of the 2A process, including a cryopreservation step, are shown. [Figure 8]

[0198] 1 shows a clinical trial design including cohorts treated with embodiments of Process 1C and Process 2A. [Figure 9]

[0199] 2 is a chart of an exemplary process 2A providing an overview of steps A through F. [Figure 10]

[0200] 1 is a process flow chart of Process 2A Data Collection Plan. [Figure 11]

[0201] 1 is a schematic of an exemplary embodiment of the Rapid Expansion Protocol (REP). Upon arrival, tumors are fragmented and placed in G-Rex flasks containing IL-2 for 11 days of TIL expansion (pre-REP expansion). For triple cocktail studies, IL-2 / IL-15 / IL-21 are added at the beginning of pre-REP. For the Rapid Expansion Protocol (REP), TILs are further cultured on feeders and OKT3 for 11 days of REP expansion. [Figure 12]

[0202] 2A shows a schematic diagram of an embodiment of Process 2A, a 22-day process for TIL fabrication. [Figure 13]

[0203] 1 is a comparison table of steps A through F from exemplary embodiments of Process 1C and Process 2A. [Figure 14]

[0204] 1 is a detailed comparison of an embodiment of Process 1C with an embodiment of Process 2A. [Figure 15]

[0205] 1 is a detailed scheme of an embodiment of a TIL therapy process. [Figure 16]

[0206] 1 is a depiction of an embodiment of a cryopreserved TIL manufacturing process (22 days). [Figure 17]

[0207] This is a table of process improvements from Gen 1 to Gen 2. [Figure 18]

[0208] 1 is an embodiment of a TIL manufacturing process of the present invention. [Figure 19a]

[0209] 1 is a process flowchart of Process 2A. [Figure 19b]

[0209] Process flow chart of Process 2A. [Figure 19c]

[0209] Process flow chart of Process 2A. [Figure 20]

[0210] 1 is a depiction of one embodiment of a TIL manufacturing process that includes an electroporation step for use in gene editing processes (including TALENs, zinc finger nucleases, and CRISPR methods described herein). [Figure 21]

[0211] 1 is a depiction of an embodiment of a TIL manufacturing process that includes an electroporation step for use in gene editing processes (including TALENs, zinc finger nucleases, and CRISPR methods described herein). [Figure 22]

[0212] In the depiction of Structures IA and IB, the cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains, e.g., derived from an antibody that binds to 4-1BBL or 4-1BB, which fold to form a trivalent protein and then link to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link the two trivalent proteins via disulfide bonds (small, elongated ovals), stabilizing the structure and providing an agonist that can combine the six receptor intracellular signaling domains and signaling proteins to form a signaling complex. The TNFRSF-binding domain depicted as a cylinder can be, for example, an scFv domain comprising VH and VL chains connected by a linker that may contain hydrophilic residues, as well as Gly and Ser sequences for flexibility and Glu and Lys for solubility. [Figure 23]

[0213] 1 is a depiction of a TALEN construct targeting exon 2 of the Pdcd1 gene. DETAILED DESCRIPTION OF THE INVENTION

[0192] Detailed Description of the Invention I. Introduction

[0214] TILs cultured ex vivo by rapid expansion protocol (REP) The use of adoptive cell therapy has resulted in successful adoptive cell therapy in melanoma patients following host immunosuppression. Current infusion eligibility parameters rely on readings of TIL composition (e.g., CD28, CD8, or CD4 positivity) and numerical values ​​of fold expansion and survival of the REP product.

[0193]

[0215] The current REP protocol does not guarantee the health of the TILs that will be infused into patients. T cells undergo a significant metabolic shift during their maturation from naive to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, hereby expressly incorporated in its entirety, and particularly for the discussion and markers of anaerobic and aerobic metabolism). For example, naive T cells rely on mitochondrial respiration for ATP production, whereas mature, healthy effector T cells, such as TILs, are highly glycolytic, relying on aerobic glycolysis to provide the bioenergetic substrates required for proliferation, migration, activation, and antitumor efficacy.

[0194]

[0216] Previous studies have shown that cells that rely heavily on glycolysis are nutrient-deprived during adoptive transfer. Because TILs undergo glycolysis and promote mitochondrial metabolism before transfer, it is desirable to limit glycolysis and promote mitochondrial metabolism before transfer. Therefore, the art teaches that promoting mitochondrial metabolism may promote in vivo lifespan, and in fact, it has been proposed to use glycolysis inhibitors before inducing an immune response. See Chang, et al., Nat.Immunol.2016, 17(364).

[0195]

[0217] The present invention, in some embodiments, determines and quantifies this increase in metabolic health. Accordingly, the present invention provides methods for assaying the relative health of a TIL population using one or more common metabolic determinations, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve.

[0196]

[0218] Moreover, the present invention, in some embodiments, provides a method for determining this increased metabolic health. Accordingly, the present invention provides methods for assaying the relative health of a TIL population using one or more common metabolic determinations, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve.

[0197]

[0219] Additionally, optional additional determinations include, but are not limited to, ATP production, These include mitochondrial mass and glucose uptake.

[0198]

[0220] The present invention provides, in some embodiments, a method for treating TILs using gene editing techniques.

[0003] It is further directed to enhancing therapeutic efficacy. While adoptive transfer of tumor-infiltrating lymphocytes (TILs) offers promising and effective therapies, there is a strong need for more effective TIL therapies that can increase patient response rates and robustness of response. As described herein, embodiments of the present invention provide methods for expanding TILs in therapeutic populations that are gene-edited to provide enhanced therapeutic efficacy.

[0199] II. Definition

[0221] Unless otherwise defined, all technical and scientific terms used herein are and have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0200]

[0222] The term "in vivo" refers to an event that takes place inside a subject's body.

[0201]

[0223] The term "in vitro" refers to events that occur outside a subject's body. The assays include cell-based assays that utilize live or dead cells, and may also include cell-free assays that do not utilize intact cells.

[0202]

[0224] The term "ex vivo" refers to cells, tissues and / or organs that have been removed from a subject's body. It refers to an event that involves the treatment or administration of a procedure on a subject. Suitably, the cells, tissues and / or organs are returned to the subject's body by way of surgery or treatment.

[0203]

[0225] The term "rapid expansion culture" refers to an expansion of at least about 3-fold (or By rapid expansion is meant an increase in the number of antigen-specific TILs of at least about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold), 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 at least about 100-fold over a one-week period. Several rapid expansion protocols are outlined below.

[0204]

[0226] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to cells that migrate through the bloodstream of a subject. "TIL" refers to a population of cells originally obtained as white blood cells that have migrated away from the tumor and into it. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations expanded or grown 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.

[0205]

[0227] As used herein, a "cell population" (including TILs) refers to a group of cells that share a common trait. refers to a large number of cells. Generally, a population is roughly 1 x 10 6 ~1×10 10 The range is 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion cultures typically yield a bulk TIL population of 1.5 x 10 cells. 9 ~1.5×10 10 This is performed to provide a population of cells for injection.

[0206]

[0228] As used herein, "cryopreserved TIL" refers to TILs obtained from primary, bulk, or expanded cultures (R Cryopreservation refers to the processing and storage of TILs (whether cryopreserved or cryopreserved TILs) at temperatures ranging from about -150°C to -60°C. General cryopreservation methods are 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.

[0207]

[0229] As used herein, "thawed cryopreserved TILs" refers to TILs that have been previously cryopreserved. By "TILs" is meant a population of TILs that has been cultured at room temperature and then treated to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs may be administered to a patient.

[0208]

[0230] TILs are generally identified biochemically using cell surface markers or by tumor immunohistochemistry. TILs can be functionally defined by their ability to infiltrate and affect treatment. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0209]

[0231] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used to cryopreserve cells. Such media can include media containing 7%-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 is sometimes referred to under the trade name "CryoStor (registered trademark) CS10." CS10 medium is a serum-free, animal component-free medium containing DMSO.

[0210]

[0232] The term "central memory T cells" refers to cells that are CD45R0+ in humans and Central memory T cells are a subset of T cells that constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of 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 are predominant in the CD4 compartment of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0211]

[0233] The term "effector memory T cells" is similar to that of central memory T cells. This refers to a subset of human or mammalian T cells that are primarily CD45R0+ but lack constitutive expression of CCR7 (CCR7lo) and have heterogeneous or low CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. After antigen stimulation, effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells have high amounts of perforin. The term "closed system" refers to a system that is closed from 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. After the tumor segments are added to the closed system, the system is not opened to the external environment until just before administration of the TILs to the patient.

[0212]

[0234] "Fragmentation" is used herein to describe the process of disrupting tumors. The terms "fragment" and "fragmented" include crushing, slicing, dividing, and fragmenting tumor tissue. Mechanical fragmentation methods such as morsel and any other method that disrupts the physical structure of tumor tissue are included.

[0213]

[0235] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to lymphocytes (T cells, B cells, N cells, etc.). It refers to peripheral blood cells with round nuclei, including PBMCs (K cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0214]

[0236] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to lymphocytes (T cells, B cells, N cells, etc.). PBMC refers to peripheral blood cells with round nuclei, including PBMCs (K cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[0215]

[0237] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody. and includes human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0216]

[0238] The term "OKT-3" (also referred to herein as "OKT3") refers to the mature T It refers to monoclonal antibodies or biosimilars or variants thereof, including human, humanized, chimeric or murine antibodies directed against the CD3 receptor in the T-cell antigen receptor of cells, including OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants thereof, conservative amino acid substitutions, glycoforms This includes commercially available forms such as muromonab or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706. Anti-CD3 antibodies include the UHCT1 clone (BioLegend, San Diego, CA, USA), also known as T3 and CD3ε. (commercially available from

[0217] [Table 1]

[0218]

[0239] The term "IL-2" (also referred to herein as "IL2") refers to interleukin-2 (IL-2). The term "IL-2" refers to the T cell growth factor known as IL-2, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosure of which is incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 is used in conjunction with aldesleukin (PROLEUKIN). (Commercially available from multiple sources at 22 million IU per single-use vial) as well as forms of recombinant IL-2 commercially available from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-209-b), and other commercially available equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2, as described herein, refers to the recombinant IL-2 produced by Nektar Therapeutics, South NKTR-214, a pegylated IL2 prodrug available from San Francisco, CA, USA, is also available. Also encompassed are pegylated forms of IL-2, including NKTR-214. Suitable NKTR-214 and pegylated IL-2 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 4902,502, the disclosures of which are incorporated herein by reference. Suitable formulations of IL-2 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.

[0219] [Table 2]

[0220]

[0240] The term "IL-4" (also referred to herein as "IL4") refers to interleukin-4 (IL-4). IL-4 refers to a cytokine known as IL-4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. When activated by IL-4, Th2 T cells subsequently In a feedback loop, IL-4 produces additional IL-4. IL-4 also stimulates B cell expansion and class II MHC expression, and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is available from ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211). and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein) The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0221]

[0241] The term "IL-7" (also referred to herein as "IL7") refers to an interleukin-7 (IL7) It refers to a glycosylated tissue-derived cytokine known as ikine-7, which is available from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor α and the common γ chain receptor, in 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 available from ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA ( Human IL-7 is commercially available from several sources, including ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, 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 shown in Table 2 (SEQ ID NO: 6).

[0222]

[0242] The term "IL-15" (also referred to herein as "IL15") refers to an interferon-like protein (IL-15). "IL-15" refers to the T cell growth factor known as IL-2, also known as IL-15, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 is a β Recombinant human IL-15 shares the γ signaling receptor subunit with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is available from 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 CYT-230-b). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0223]

[0243] The term "IL-21" (also referred to herein as "IL21") refers to an interleukin-21 (IL-21) "IL-21" refers to the pleiotropic cytokine protein known as IL-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 primarily stimulates natural killer T cells and activated human CD4+ Recombinant human IL-21 is produced by T cells. It is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is available from ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog no. CY T-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0224]

[0244] When an "anti-tumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, The exact dosage of the compositions of the present invention will be determined by the physician depending on the patient's age, weight, tumor size, degree of infection or metastasis, etc. The dosage can be determined taking into consideration individual differences in the condition of the patient (subject). Generally, the pharmaceutical composition containing the tumor-infiltrating lymphocytes (e.g., secondary TIL or genetically modified cytotoxic lymphocytes) described herein is administered in a dose of 100 mg / kg or more. 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 can be said that tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocyte) compositions can be administered at a dosage of 1000 mg / kg body weight (including all integer values ​​within these ranges). Tumor-infiltrating lymphocyte (optionally including genetically modified cytotoxic lymphocytes) compositions can also be administered multiple times at these dosages. Tumor-infiltrating lymphocytes (including genetically modified ones in some cases) can be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient may be determined. Dimen can be readily determined by one of ordinary skill in the art of medicine by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0225]

[0245] The term "hematologic malignancies" refers to tumors of the blood, bone marrow, lymph nodes, and tissues of the lymphatic system. The term "B-cell hematological malignancies" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0226]

[0246] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid 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, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells), and supporting stromal cells in which cancer cells may be dispersed and provide a supportive microenvironment.

[0227]

[0247] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs obtained from liquid tumors are also referred to herein as bone marrow infiltrating lymphocytes (MILs).

[0228]

[0248] As used herein, the term "microenvironment" refers to a solid or The term "tumor microenvironment" may refer to the blood tumor microenvironment or individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment is defined as described in Swartz, et al., Cancer Res., 2012, 72, 2473. As described by the National Cancer Institute (NCI), tumor necrosis factor (TNF) refers to a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, cultivate therapeutic resistance, and provide a niche for successful and dominant metastasis. Although tumors express antigens that are recognized by T cells, tumor elimination by the immune system is rare due to the immunosuppressive microenvironment.

[0229]

[0249] In one embodiment, the invention includes a method of treating cancer with a TIL population, In some embodiments, a TIL population can be provided, wherein the patient is conditioned with non-myeloablative chemotherapy prior to the infusion of the TILs of the present invention. In one embodiment, the non-myeloablative chemotherapy comprises cyclophosphamide 60 mg / kg / day for two days (27 and 26 days prior to the TIL infusion) and fludarabine 25 mg / m 2 for five days (27-23 days prior to the TIL infusion). 2 / In one embodiment, after non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), patients receive an intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours to a physiologically tolerated dose.

[0230]

[0250] Experimental findings suggest that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes may modulate These findings suggest that rTILs play an important role in enhancing the efficacy of treatment by eliminating competing components of the immune system ("cytokine sinks") and T cells. Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") on patients prior to administering the rTILs of the present invention.

[0231]

[0251] As used herein, "co-administration," "co-administering," and "combination with" The terms "administered in combination with," "administering in combination with," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., at least one potassium channel agonist in combination with multiple TILs) 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.

[0232]

[0252] The term "effective amount" or "therapeutically effective amount" includes, but is not limited to, disease treatment. The term "therapeutically effective" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, without affecting the intended therapeutic effect. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo) or the subject and disease state being treated (e.g., the subject's weight, age, and sex), the severity of the disease state, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., decreased platelet adhesion and / or cell migration). The specific dose 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 by which the compound is delivered.

[0233]

[0253] The terms "treatment," "treating," "treat," and the like refer to the desired pharmacological and "Treatment" refers to achieving a specific and / or physiological effect. The effect may be prophylactic, meaning that the disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that the disease and / or adverse effects resulting from the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the occurrence of the disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., halting 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 intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a disease state, e.g., in the case of a vaccine.

[0234]

[0254] The term "heterologous" when used in reference to a nucleic acid or protein moiety indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). Protein).

[0235]

[0255] In the context of two or more nucleic acids or polypeptides, "sequence identity," "percent The terms "sequence identity" and "percent sequence identity" (or their synonyms, e.g., "99% identical") refer to two or more sequences or subsequences that are the same or 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 the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST program available from the BLAST website of the U.S. government's National Center for Biotechnology Information. The BLAST suite of programs is a useful tool for aligning sequences. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Publicly available programs such as ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR can be used to align sequences. Further software programs that can be used to align sequences are also available. Those skilled in the art can determine appropriate parameters for maximal alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0236]

[0256] As used herein, the term "variant" refers to a variant within the amino acid sequence of a reference antibody. The term "variant" includes, but is not limited to, antibodies or fusion proteins comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions in or adjacent to the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of amino acids that are similarly charged or uncharged. A variant retains the ability of the reference antibody to specifically bind to the antigen. The term "variant" also includes pegylated antibodies or proteins.

[0237]

[0257] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to cells that migrate through the bloodstream of a subject. "TILs" refers to a population of cells originally obtained as white blood cells that have migrated away from the tumor and into it. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary 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 expanded or grown 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., those described in step D of FIG. 9 , which include TILs designated as reREP TILs).

[0238]

[0258] TILs are generally identified biochemically using cell surface markers or by tumor immunohistochemistry. TILs can be functionally defined by their ability to infiltrate and affect treatment. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency - for example, TILs can be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL.

[0239]

[0259] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a This includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or 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.

[0240]

[0260] The terms "about" and "approximately" refer to within a statistically significant range of values. The ranges 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 allowable variation encompassed by the term "about" or "approximately" depends on the particular system under study and is readily apparent to one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement error, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not explicitly stated as such. It should be noted that embodiments of widely differing sizes, shapes, and dimensions may refer to the described terms.

[0241]

[0261] As used in the appended claims, the terms "comprising," "consisting essentially of" and "composing" are used interchangeably. The transitional terms "comprising" and "consisting of" define the claims in their original and amended form, with respect to the exclusion from the claim of any additional unrecited claim elements or steps, if any. 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 in the latter case, excludes normal impurities associated with the specified material. The term "consisting essentially of" limits the claim to the specified elements, steps, or materials and those that do not materially affect the basic and novel characteristics of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "essentially consisting of," and "consisting of."

[0242] III. Gene Editing Process A. Overview: TIL expansion and gene editing

[0262] Embodiments of the present invention are directed to methods for expanding a population of TILs, the methods comprising: The method includes one or more steps of gene editing at least a portion of the TILs to enhance its therapeutic effect. As used herein, "gene-editing," "gene editing," and "genome editing" refer to a type of genetic modification in which DNA is permanently altered in the genome of a cell, e.g., DNA is inserted, deleted, modified, or replaced within the genome of a cell. In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockdown) expression of a DNA sequence. In other embodiments, gene editing enhances expression of a DNA sequence (e.g., by causing overexpression). EMBODIMENTS OF THE INVENTION According to one embodiment, gene editing techniques are used to enhance the efficacy of therapeutic TIL populations.

[0243]

[0263] The method for expanding tumor-infiltrating lymphocytes (TILs) into therapeutic TIL populations is described in this paper. The method may be performed according to any embodiment of the methods described herein (e.g., an exemplary TIL expansion method known as Process 2A is described below), where the method includes gene editing at least a portion of the TILs. According to additional embodiments, the method of expanding TILs into a therapeutic TIL population is performed according to any embodiment of the methods described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633 (which are incorporated by reference herein in their entireties), where the method includes gene editing at least a portion of the TILs. Thus, one embodiment of the present invention provides a therapeutic TIL population expanded according to any embodiment described herein, where at least a portion of the therapeutic population has been gene edited, e.g., at least a portion of the therapeutic TIL population transferred to an infusion bag has been permanently gene edited.

[0244] B. Timing of gene editing during TIL expansion

[0264] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture to generate a second TIL population by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 (e.g., OKT-3 can be present in the culture medium starting on the initiation day of the expansion culture process), wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; (f) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; and (g) gene editing at least a portion of the TILs at any point during the method. Includes.

[0245]

[0265] As noted in step (g) of the above embodiment, the gene editing process comprises: Gene editing can be performed at any point during the TIL expansion method, meaning that gene editing can be performed on the TILs before, during, or after any step of the expansion method (e.g., during any of steps (a)-(f) outlined in the method above, or before or after any of steps (a)-(f) outlined in the method above). According to certain embodiments, the TILs are collected during the expansion method (e.g., the expansion method is "paused" for at least some of the TILs), and the collected TILs are subjected to the gene editing process, possibly In some cases, the TILs are then reintroduced into the expansion method (e.g., returned to culture medium) to continue the proliferation process, resulting in permanent gene editing of at least a portion of the therapeutic TIL population that is ultimately transferred to the infusion bag. In one embodiment, the gene editing process can be carried out prior to expansion by activating the TILs, performing a gene editing step on the activated TILs, and expanding the gene-edited TILs according to the processes described herein.

[0246]

[0266] Alternative embodiments of the expansion process may differ from the method shown above. Note that, for example, alternative embodiments may not have the same steps (a)-(g), or may have a different number of steps. Regardless of the particular embodiment, the gene editing process may be performed at any time during the TIL expansion method. For example, alternative embodiments may include three or more expansions, and gene editing may be performed on the TILs during the third or fourth expansion, etc.

[0247]

[0267] According to one embodiment, the gene editing process comprises: The gene editing is performed on TILs from one or more of the third populations. For example, gene editing can be performed on the first TIL population or a portion of the TILs collected from the first population, and after the gene editing process, the TILs can then be returned to the expansion process (e.g., returned to the medium). Alternatively, gene editing can be performed on TILs from the second or third population, or a portion of the TILs collected from the second or third population, respectively, and after the gene editing process, the TILs can then be returned to the expansion process (e.g., returned to the medium). According to another embodiment, gene editing is performed while the TILs are still in the medium and expansion is being carried out. That is, they are not necessarily "removed" from the expansion culture to perform gene editing.

[0248]

[0268] According to another embodiment, the gene editing process comprises the step of: Gene editing can be performed on TILs from the first or second expansion culture, or both. For example, during the first or second expansion culture, gene editing can be performed on TILs collected from the culture medium, and following the gene editing process, the TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium.

[0249]

[0269] According to another embodiment, the gene editing process is carried out after the first expansion and after the second expansion. For example, after a first expansion, gene editing can be performed on TILs collected from the culture medium, and following the gene editing process, the TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium for a second expansion.

[0250]

[0270] According to an alternative embodiment, before step (c) (e.g., steps (a) to ( The gene editing process is performed before step (d) (e.g., before, during or after any of steps (a)-(d)), before step (d) (e.g., before, during or after any of steps (a)-(d)), or before step (f) (e.g., before, during or after any of steps (a)-(e)).

[0251]

[0271] Regarding OKT-3, according to certain embodiments, on day 0 and / or day 1 It should be noted that the cell culture medium may contain OKT-3 starting on the start day (day 0) or day 1 of the first expansion culture, such that gene editing is performed on the TILs after exposure to OKT-3 in the cell culture medium. According to another embodiment, the cell culture medium contains OKT-3 during the first expansion culture and / or the second expansion culture, and gene editing is performed before OKT-3 is introduced into the cell culture medium. Alternatively, the cell culture medium may contain OKT-3 during the first expansion culture and / or the second expansion culture. OKT-3 may be included in the first and / or second expansion cultures, and gene editing may be performed after OKT-3 is introduced into the cell culture medium.

[0252]

[0272] With respect to 4-1BB agonists, according to certain embodiments, day 0 and / or It should also be noted that the cell culture medium can contain the 4-1BB agonist starting on the start day (day 0) of the first expansion culture or on day 1, such that gene editing is performed on the TILs after they have been exposed to the 4-1BB agonist in the cell culture medium on day 1. According to another embodiment, the cell culture medium contains the 4-1BB agonist during the first expansion culture and / or the second expansion culture, and gene editing is performed before the 4-1BB agonist is introduced into the cell culture medium. Alternatively, the cell culture medium can contain the 4-1BB agonist during the first expansion culture and / or the second expansion culture, and gene editing is performed after the 4-1BB agonist is introduced into the cell culture medium.

[0253]

[0273] Regarding IL-2, according to certain embodiments, cells are administered on day 0 and / or day 1. It should also be noted that the cell culture medium can include IL-2 starting on the start day (day 0) or day 1 of the first expansion culture, such that gene editing is performed on the TILs after they have been exposed to IL-2 in the cell culture medium. According to another embodiment, the cell culture medium includes IL-2 during the first expansion culture and / or the second expansion culture, and gene editing is performed before IL-2 is introduced into the cell culture medium. Alternatively, the cell culture medium can include IL-2 during the first expansion culture and / or the second expansion culture, and gene editing is performed after IL-2 is introduced into the cell culture medium.

[0254]

[0274] As described above, one or more of OKT-3, a 4-1BB agonist, and IL-2 may be included in the cell culture medium starting on day 0 or day 1 of the first expansion culture. According to one embodiment, OKT-3 is included in the cell culture medium starting on day 0 or day 1 of the first expansion culture, and / or a 4-1BB agonist is included in the cell culture medium starting on day 0 or day 1 of the first expansion culture, and / or IL-2 is included in the cell culture medium starting on day 0 or day 1 of the first expansion culture. According to one example, the cell culture medium comprises OKT-3 and a 4-1BB agonist starting on day 0 or day 1 of the first expansion culture. According to another example, the cell culture medium comprises OKT-3, a 4-1BB agonist, and IL-2 starting on day 0 or day 1 of the first expansion culture. Of course, as described in various embodiments herein, one or more of OKT-3, a 4-1BB agonist, and IL-2 may be added to the cell culture medium at one or more further time points during the expansion culture process.

[0255]

[0275] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, the sterile electroporation step comprising: Mediating and carrying out transfers; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (g) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. Includes.

[0256]

[0276] According to one embodiment, the aforementioned method is used to treat a human subject with cancer. Autologous harvested TIL populations can be provided for this purpose.

[0257] C. Immune Checkpoints

[0277] According to certain embodiments of the present invention, the TIL population comprises one Gene editing is achieved by genetically modifying the above immune checkpoint genes. In other words, the DNA sequence in the TIL that encodes one or more of the TIL's immune checkpoints is permanently modified, e.g., inserted, deleted, or substituted, in the TIL's genome. Immune checkpoints are molecules expressed by lymphocytes that regulate immune responses via inhibitory or stimulatory pathways. In cancer, immune checkpoint pathways are activated to inhibit anti-tumor responses; i.e., expression of specific immune checkpoints by malignant cells often inhibits anti-tumor immunity and favors cancer cell proliferation. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39. Therefore, specific inhibitory checkpoints The immune molecules serve as targets for immunotherapy of the present invention. According to certain embodiments, TILs are gene-edited to block or stimulate specific immune checkpoint pathways, thereby enhancing the body's immunological activity against tumors.

[0258]

[0278] As used herein, immune checkpoint genes are genes that are involved in immune checkpoint The therapeutic TILs contain a DNA sequence encoding an immune checkpoint molecule. According to certain embodiments of the present invention, gene editing of TILs during the TIL expansion method causes silencing or reduction in the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. For example, gene editing can cause silencing or reduction in the expression of inhibitory receptors such as PD-1 or CTLA-4 to enhance the immune response.

[0259]

[0279] The most widely studied checkpoints include programmed cell death receptors (PDCRs). These include PD-1 (PD-1) and cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4), which are inhibitory receptors on immune cells that inhibit key effector functions (e.g., activation, proliferation, cytokine release, cytotoxicity, etc.) when they interact with inhibitory ligands. In addition to PD-1 and CTLA-4, numerous checkpoint molecules have emerged as potential targets for immunotherapy, as described in more detail below.

[0260]

[0280] The present invention aims to silence or inhibit TILs by permanent gene editing. Non-limiting examples of immune checkpoint genes that can be disrupted include PD-1, CTLA-4, LAG-3, HAVCR2(TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, BA FF(BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, C These include ASP7, 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, ANKRD11, SOCS1, 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, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018 (in press). According to another example, the present invention Immune checkpoint genes that may be silenced or inhibited in TILs may be selected from the group including PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0261]

[0281] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. The electroporation step includes PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3 ), and combinations thereof.

[0262] 1.PD-1

[0282] One of the most studied targets for induction of checkpoint blockade is T The programmed death receptor (PD1 or PD-1, also known as PDCD1) is a member of the CD28 superfamily of cellular regulators. Its ligands, PD-L1 and PD-L2, are expressed on a variety of tumor cells, including melanoma. PD-1 and PD-L1 interaction inhibits T cell effector function, causes T cell exhaustion in the case of chronic stimulation, and induces T cell apoptosis in the tumor microenvironment. PD1 may also play a role in tumor-specific escape from immune surveillance.

[0263]

[0283] According to a particular embodiment, the expression of PD1 in TILs is determined by the composition of the present invention. and silenced or reduced according to the method. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method includes gene editing at least a portion of the TILs by silencing or suppressing expression of PD1. As described in more detail below, the gene editing process can involve the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as PD1. For example, CRISPR, TALE, or zinc finger methods can be used to silence or reduce PD1 expression in TILs.

[0264] 2.CTLA-4

[0284] CTLA-4 expression is induced upon T cell activation in activated T cells CTLA-4 competes for binding with antigen-presenting cell activation antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 causes T cell inhibition and helps maintain a balance in the immune response. However, inhibiting the interaction of CTLA-4 with CD80 or CD86 can prolong T cell activation and increase the level of immune responses to cancer antigens.

[0265]

[0285] According to a particular embodiment, the expression of CTLA-4 in TILs is Silenced or reduced according to compositions and methods. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing the expression of CTLA-4. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as CTLA-4. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of CTLA-4 in TILs.

[0266] 3. LAG-3

[0286] Lymphocyte activation gene-3 (LAG-3, CD223) is a major histocompatibility complex It is expressed by T cells and natural killer (NK) cells after complex (MHC) class II ligation. Although the mechanism remains unclear, its regulation causes a negative regulatory effect on T cell function, preventing tissue damage and autoimmunity. LAG-3 and PD-1 are frequently co-expressed and upregulated on TILs, leading to immune exhaustion and tumor growth. Thus, LAG-3 blockade improves anti-tumor responses. See, e.g., Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

[0267]

[0287] According to a particular embodiment, the expression of LAG-3 in TILs is The expression of LAG-3 in TILs can be silenced or reduced according to the compositions and methods. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing the expression of LAG-3. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as LAG-3. According to certain embodiments, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of LAG-3 in TILs.

[0268] 4. TIM-3

[0288] T cell immunoglobulin-3 (TIM-3) is a direct negative regulator of T cells TIM-3 is a marker expressed on NK cells and macrophages. It indirectly promotes immunosuppression by inducing the expansion of myeloid-derived suppressor cells (MDSCs). Its levels have been found to be particularly elevated in dysfunctional and exhausted T cells, suggesting an important role in malignancies.

[0269]

[0289] According to a particular embodiment, the expression of TIM-3 in TILs is The expression of TIM-3 in immune checkpoint genes can be silenced or reduced according to the compositions and methods. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing the expression of TIM-3. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as TIM-3. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of TIM-3 in TILs.

[0270] 5. Cish

[0290] Ci, a member of the suppressor of cytokine signaling (SOCS) family Cish is induced by TCR stimulation of CD8+ T cells and inhibits their functional affinity for tumors. Genetic deletion of Cish in CD8+ T cells enhances their expansion, functional affinity, and cytokine multifunction, and can result in significant and durable regression of established tumors. See, e.g., Palmer et al., Journal of Experimental Medicine, 212 (12):2095 (2015).

[0271]

[0291] According to certain embodiments, expression of Cish in TILs is achieved by the use of compositions of the present invention. The expression of Cish can be silenced or reduced according to the methods and methods. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing expression of Cish. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as Cish. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress expression of Cish in TILs.

[0272] 6. TGFβ

[0292] The TGFβ signaling pathway regulates cell proliferation, differentiation, apoptosis, motility, and TGFβ has multiple functions in regulating tumor invasion, extracellular matrix production, angiogenesis, and immune response. Deregulation of TGFβ signaling frequently occurs in tumors and plays a crucial role in tumor initiation, development, and metastasis. At the microenvironment level, the TGFβ pathway contributes to the generation of a favorable microenvironment for tumor growth and metastasis throughout carcinogenesis. For example, Neuzillet et al. al., Pharmacology & Therapeutics, Vol. 147, pp. 22-31 (2015).

[0273]

[0293] According to a particular embodiment, the expression of TGFβ in TILs is regulated by the composition of the present invention. The expression of TGFβ can be silenced or reduced according to the methods and methods. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or reducing the expression of TGFβ. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as TGFβ. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of TGFβ in TILs.

[0274]

[0294] In some embodiments, TGFβR2 (TGF beta receptor 2) is CR TGFβR2 can be silenced using the ISPR / Cas9 system or by using a TGFβR2 dominant-negative extracellular trap using methods known in the art.

[0275] 7. PKA

[0295] Protein kinase A (PKA) is a serine-threonine protein kinase PKA is a well-known member of the kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multiunit protein kinase that mediates signal transduction of G protein-coupled receptors through its activation upon cAMP binding. It is involved in the regulation of diverse cellular processes, from metabolism to ion channel activation, cell proliferation and differentiation, gene expression, and apoptosis. Importantly, PKA has been implicated in the initiation and progression of many tumors. See, e.g., Sapio et al., EXCLI Journal; 2014; 13:843-855. Please refer to.

[0276]

[0296] According to a particular embodiment, the expression of PKA in TILs is regulated by the composition of the present invention. and silenced or reduced according to the method. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method includes gene editing at least a portion of the TILs by silencing or suppressing expression of PKA. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as PKA. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress expression of PKA in TILs.

[0277] 8.CBLB

[0297] CBLB (or CBL-B) is an E3 ubiquitin protein ligase , a negative regulator of T cell activation. Bachmaier, et al., Nature, 2000, 403, 211-216; Wallner, et al., Clin. Dev. Immunol. 2012, 692-639. The disclosures of which are further incorporated by reference. In U.S. Patent Application Publication No. 2019 / 0284530 (the '530 publication), which is incorporated herein, reducing CBLB expression or function is shown to improve immunotherapy by enhancing the effector function of immune effector cells. Examples 12, 13, 14, and 16 of the '530 publication demonstrate various embodiments of CBLB modulation in TILs and other cells.

[0278]

[0298] According to a particular embodiment, the expression of CBLB in TILs is determined by the composition of the present invention. The expression of PKA in TILs can be silenced or reduced according to the methods and methods described herein. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing the expression of CBLB. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as CBLB. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of PKA in TILs. In some embodiments, CBLB is silenced using TALEN knockout. In some embodiments, CBLB is silenced using TALE-KRAB transcription inhibitor knockin. Details of these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585.

[0279] 9. TIGIT

[0299] Contains Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains The T cell immunoreceptor (TIGIT), or TIGIT-dependent T cell immunoreceptor (TIGIT), is a transmembrane glycoprotein receptor with an Ig-like V-type domain and ITIM in its cytoplasmic domain. Khalil, et al., Advances in Cancer Research, 2015, 128, 1-68; Yu, et al., Nature Immunology, 2009, Vol. 10, No. 1, 48-57. TIGIT is expressed by a subset of T cells and natural killer cells. Furthermore, TIGIT has been shown to be overexpressed in antigen-specific CD8+ T cells and CD8+ TILs, particularly from individuals with melanoma. Studies have shown that the TIGIT pathway contributes to tumor immune evasion, and that TIGIT inhibition increases T cell activation and proliferation in response to polyclonal and antigen-specific stimulation. Khalil, et al., Advances in Cancer Research, 2015, 128, 1-68. Furthermore, co-blockade of TIGIT with PD-1 or TIM3 has shown synergistic effects against solid tumors in mouse models. See also The Journal of Clinical Investigation, 2015, Vol. 125, No. 11, 4053-4062.

[0280]

[0300] According to a particular embodiment, the expression of TIGIT in TILs is The expression of TIGIT can be silenced or reduced according to the compositions and methods. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing the expression of TIGIT. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as TIGIT. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of TIGIT in TILs.

[0281] 10. TOX

[0301] Thymocyte selection-associated high mobility group (HMG) box (TOX) is a thymocyte selection-associated high mobility group (HMG) box TOX is a transcription factor containing a TOX DNA-binding domain. TOX is a member of the HMG box superfamily and binds to DNA in a sequence-independent but structure-dependent manner. It is believed that...

[0282]

[0302] For example, as described in Scott, et al., Nature, 2019, 571, 270-274 and Khan, et al., Nature, 2019, 571, 211-218 (both of which are incorporated herein by reference in their entirety), TOX inhibits tumor-specific CD8 + CD8 has been identified as a key regulator of T cell dysfunction or T cell exhaustion and is transcriptionally and epigenetically + TOX was found to program T cell exhaustion. TOX inhibits T cell function during chronic infection, as described in Alfei, et al., Nature, 2019, 571, 265-269, the entire contents of which are incorporated herein by reference. TOX has also been found to be an important factor in the progression of disease and the maintenance of exhausted T cells. TOX is highly expressed in T cells that are dysfunctional or exhausted due to tumors and chronic viral infections. Ectopic expression of TOX in effector T cells in vitro induced the transcriptional program associated with T cell exhaustion, whereas deletion of TOX in T cells abolished the T cell exhaustion program.

[0283]

[0303] According to a particular embodiment, the expression of TOX in TILs is determined by the composition of the present invention. and silenced or reduced according to the method. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing expression of TOX. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-stranded breaks in immune checkpoint genes such as TOX. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress TOX expression in TILs.

[0284] 11.ANKRD11

[0304] The ANKRD11 gene is an ankyrin repeat domain 11 (ANKRD1 ANKRD11 encodes a protein called nasopharyngeal carcinoma susceptibility protein (NAPC), sometimes referred to as nasopharyngeal carcinoma susceptibility protein. This protein contains multiple ankyrin domains, which facilitate interactions between other proteins. ANKRD11 has been shown to be downregulated in breast cancer and to be associated with breast tumorigenesis. This downregulation of ANKRD11 may be associated with promoter DNA methylation (Lim, et al., Eur. J. Cancer, 2012, 48(17):3300-3309).

[0285]

[0305] Further, in the '530 publication, the disclosure of which is incorporated herein by reference: Reducing ANKRD11 expression or function has been shown to improve immunotherapy by enhancing the effector function of immune effector cells. Examples 7, 15, 16, 18-21, 27, and 30 of the '530 publication demonstrate various embodiments of ANKRD11 modulation in TILs and other cells.

[0286]

[0306] According to a particular embodiment, the expression of ANKRD11 in TILs is The expression of ANKRD11 is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing the expression of ANKRD11. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as ANKRD11. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of ANKRD11 in TILs.

[0287] 12.SOCS1

[0307] The SOCS1 gene is the suppressor of cytokine signaling 1 protein SOCS1 encodes SOCS1, which is involved in a negative feedback loop to attenuate JAK-STAT cytokine signaling and inhibit inflammation (Sharma, et al., Front.Pharmacol.2019, doi.org / 10.3389 / fphar.2019.00324). Research suggests that this protein is a regulator of IFN-γ. SOCS1 is also recognized as a tumor suppressor in various cancers, and there is evidence that SOCS1 is an oncogene, as described, for example, in Gui et al., Oncogene, 2016, 35(32):4200-4211.

[0288]

[0308] Furthermore, the '530 publication states that a decrease in SOCS1 expression or function is associated with an immune response. These studies have been shown to improve immunotherapy by enhancing the effector function of effector cells. Examples 7, 14, 16, 23-32 of the '530 publication demonstrate various embodiments of SOCS1 modulation in TILs and other cells.

[0289]

[0309] According to a particular embodiment, the expression of SOCS1 in TILs is The expression of SOCS1 can be silenced or reduced according to the compositions and methods. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or reducing the expression of SOCS1. For example, CRISPR, TALE, or zinc finger techniques can be used to silence or reduce the expression of SOCS1 in TILs.

[0290] 13.BCOR

[0310] BCL6 corepressor (BCOR) is a protein that regulates the function of BCL-6 corepressor proteins. BCOR is a gene encoding a protein. This gene is a member of the ankyrin repeat domain containing gene family. This protein is a POZ / zinc finger transcriptional repressor required for germinal center formation and may affect apoptosis. BCOR is known to be an epigenetic regulator and has been increasingly identified as mutated in various human cancers (Astolfi et al., Epigenomics, 2019, 11(7):835-855).

[0291]

[0311] Furthermore, in the '530 publication, a decrease in BCOR expression or function was found to be associated with immune responses. These methods have been shown to improve immunotherapy by enhancing the effector function of TIL and other cells. Examples 12 and 13 of the '530 publication demonstrate various embodiments of BCOR modulation in TIL and other cells.

[0292]

[0312] According to a specific embodiment, the expression of BCOR in TILs is regulated by the composition of the present invention. The expression of BCOR can be silenced or reduced according to the methods and methods. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., Process 2A or the methods shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by silencing or suppressing BCOR expression. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes such as BCOR. For example, CRISPR, TALE, or zinc finger techniques can be used to silence or suppress BCOR expression in TILs.

[0293]

[0313] In another embodiment, the present invention provides a method for stimulating the expression of PD-1 and CTLA-4.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0294]

[0314] In another embodiment, the present invention provides a method for silencing PD-1 and LAG-3 expression.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0295]

[0315] In another embodiment, the present invention provides a method for silencing PD-1 and TIM-3 expression.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0296]

[0316] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and Cish.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0297]

[0317] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and TGFβ.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0298]

[0318] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and PKA.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0299]

[0319] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and CBLB.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0300]

[0320] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0301]

[0321] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0302]

[0322] In another embodiment, the present invention provides a method for detecting PD-1 and ANKRD11 expression.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0303]

[0323] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0304]

[0324] In another embodiment, the present invention provides a method for silencing the expression of PD-1 and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0305]

[0325] In another embodiment, the present invention provides a method for inhibiting the expression of CTLA-4 and LAG-3.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0306]

[0326] In another embodiment, the present invention provides a method for inhibiting the expression of CTLA-4 and TIM-3.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0307]

[0327] In another embodiment, the present invention provides a method for stimulating the expression of CTLA-4 and Cish.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0308]

[0328] In another embodiment, the present invention provides a method for stimulating the expression of CTLA-4 and TGFβ.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0309]

[0329] In another embodiment, the present invention provides a method for silencing the expression of CTLA-4 and PKA.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0310]

[0330] In another embodiment, the present invention provides a method for stimulating the expression of CTLA-4 and CBLB.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0311]

[0331] In another embodiment, the present invention provides a method for inhibiting the expression of CTLA-4 and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0312]

[0332] In another embodiment, the present invention provides a method for silencing the expression of CTLA-4 and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0313]

[0333] In another embodiment, the present invention relates to a method for detecting the expression of CTLA-4 and ANKRD11. In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing

[0314]

[0334] In another embodiment, the present invention provides a method for inhibiting the expression of CTLA-4 and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0315]

[0335] In another embodiment, the present invention provides a method for stimulating the expression of CTLA-4 and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0316]

[0336] In another embodiment, the present invention provides a method for stimulating the expression of LAG-3 and TIM-3.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0317]

[0337] In another embodiment, the present invention provides a method for silencing the expression of LAG-3 and Cish.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0318]

[0338] In another embodiment, the present invention provides a method for silencing the expression of LAG-3 and TGFβ.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0319]

[0339] In another embodiment, the present invention provides a method for silencing the expression of LAG-3 and PKA.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0320]

[0340] In another embodiment, the present invention provides a method for silencing the expression of LAG-3 and CBLB.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0321]

[0341] In another embodiment, the present invention provides a method for stimulating the expression of LAG-3 and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0322]

[0342] In another embodiment, the present invention provides a method for silencing the expression of LAG-3 and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0323]

[0343] In another embodiment, the present invention relates to a method for detecting the expression of LAG-3 and ANKRD11. The method of any of the preceding paragraphs above, as applicable, is provided, modified to include gene editing at least a portion of the TILs by silencing or suppressing them.

[0324]

[0344] In another embodiment, the present invention provides a method for stimulating the expression of LAG-3 and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0325]

[0345] In another embodiment, the present invention provides a method for silencing the expression of LAG-3 and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0326]

[0346] In another embodiment, the present invention provides a method for silencing the expression of TIM-3 and Cish.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0327]

[0347] In another embodiment, the present invention provides a method for silencing the expression of TIM-3 and TGFβ.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0328]

[0348] In another embodiment, the present invention provides a method for silencing the expression of TIM-3 and PKA.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0329]

[0349] In another embodiment, the present invention provides a method for silencing the expression of TIM-3 and CBLB. and gene editing at least some of the TILs by sensitizing or suppressing the TILs. The method of any of the preceding paragraphs, as appropriate, is provided, as modified as follows:

[0330]

[0350] In another embodiment, the present invention provides a method for stimulating the expression of TIM-3 and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0331]

[0351] In another embodiment, the present invention provides a method for silencing the expression of TIM-3 and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0332]

[0352] In another embodiment, the present invention provides a method for detecting the expression of TIM-3 and ANKRD11. The method of any of the preceding paragraphs above, as applicable, is provided, modified to include gene editing at least a portion of the TILs by silencing or suppressing them.

[0333]

[0353] In another embodiment, the present invention provides a method for stimulating the expression of TIM-3 and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0334]

[0354] In another embodiment, the present invention provides a method for silencing the expression of TIM-3 and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0335]

[0355] In another embodiment, the present invention provides a method for silencing the expression of Cish and TGFβ.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0336]

[0356] In another embodiment, the present invention provides a method for silencing expression of Cish and PKA.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0337]

[0357] In another embodiment, the present invention provides a method for silencing expression of Cish and CBLB.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0338]

[0358] In another embodiment, the present invention provides a method for silencing the expression of Cish and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0339]

[0359] In another embodiment, the present invention provides a method for silencing expression of Cish and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0340]

[0360] In another embodiment, the present invention provides a method for the detection of Cish and ANKRD11.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0341]

[0361] In another embodiment, the present invention provides a method for silencing the expression of Cish and SOCS1. and gene editing at least some of the TILs by sensitizing or suppressing the TILs. The method of any of the preceding paragraphs, as appropriate, is provided, as modified as follows:

[0342]

[0362] In another embodiment, the present invention provides a method for silencing the expression of Cish and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0343]

[0363] In another embodiment, the present invention provides a method for silencing the expression of TGFβ and PKA.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0344]

[0364] In another embodiment, the present invention provides a method for silencing the expression of TGFβ and CBLB.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0345]

[0365] In another embodiment, the present invention provides a method for silencing the expression of TGFβ and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0346]

[0366] In another embodiment, the present invention provides a method for silencing the expression of TGFβ and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0347]

[0367] In another embodiment, the present invention provides a method for inhibiting the expression of TGFβ and ANKRD11.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0348]

[0368] In another embodiment, the present invention provides a method for silencing the expression of TGFβ and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0349]

[0369] In another embodiment, the present invention provides a method for silencing the expression of TGFβ and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0350]

[0370] In another embodiment, the present invention provides a method for silencing the expression of PKA and CBLB.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0351]

[0371] In another embodiment, the present invention provides a method for silencing the expression of PKA and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0352]

[0372] In another embodiment, the present invention provides a method for silencing the expression of PKA and TOX.

[0023] In some embodiments, the method of claim 1 further comprises: (a) selecting a gene for at least one TIL from a plurality of TILs; (b) selecting a gene for at least one TIL from a plurality of TILs; (c) selecting a gene for at least one TIL from a plurality of TILs; and (d) selecting a gene for at least one TIL from a plurality of TILs from a plurality of TILs.

[0353]

[0373] In another embodiment, the present invention provides a method for stimulating the expression of PKA and ANKRD11.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0354]

[0374] In another embodiment, the present invention provides a method for silencing the expression of PKA and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0355]

[0375] In another embodiment, the present invention provides a method for silencing the expression of PKA and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0356]

[0376] In another embodiment, the present invention provides a method for silencing the expression of CBLB and TIGIT.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0357]

[0377] In another embodiment, the present invention provides a method for silencing expression of CBLB and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0358]

[0378] In another embodiment, the present invention provides a method for detecting CBLB and ANKRD11 expression.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0359]

[0379] In another embodiment, the present invention provides a method for silencing the expression of CBLB and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0360]

[0380] In another embodiment, the present invention provides a method for silencing the expression of CBLB and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0361]

[0381] In another embodiment, the present invention provides a method for silencing the expression of TIGIT and TOX.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0362]

[0382] In another embodiment, the present invention relates to a method for controlling the expression of TIGIT and ANKRD11. The method of any of the preceding paragraphs above, as applicable, is provided, modified to include gene editing at least a portion of the TILs by silencing or suppressing them.

[0363]

[0383] In another embodiment, the present invention provides a method for stimulating the expression of TIGIT and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0364]

[0384] In another embodiment, the present invention provides a method for silencing the expression of TIGIT and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0365]

[0385] In another embodiment, the present invention provides a method for stimulating the expression of TOX and ANKRD11.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least a portion of the TILs by suppressing or inhibiting the TILs.

[0366]

[0386] In another embodiment, the present invention provides a method for silencing the expression of TOX and SOCS1.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least a portion of the TILs by silencing or suppressing the TILs.

[0367]

[0387] In another embodiment, the present invention provides a method for silencing the expression of TOX and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by inhibiting or suppressing the TILs.

[0368]

[0388] In another embodiment, the present invention relates to a method for detecting the expression of ANKRD11 and SOCS1. The method of any of the preceding paragraphs above, as applicable, is provided, modified to include gene editing at least a portion of the TILs by silencing or suppressing them.

[0369]

[0389] In another embodiment, the present invention provides a method for inhibiting the expression of ANKRD11 and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing at least some of the TILs by silencing or suppressing the TILs.

[0370]

[0390] In another embodiment, the present invention provides a method for silencing the expression of SOCS1 and BCOR.

[0023] In some embodiments, the method is as described in any of the preceding paragraphs above, modified to include gene editing of at least some of the TILs by detecting or suppressing the TILs.

[0371]

[0391] In another embodiment, the present invention relates to a method for detecting PD-1, CTLA-4, LAG-3, HAVCR2(TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, BAFF(BR3), CD96, CRTAM, LAIR1, S IGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SK The method of any of the preceding paragraphs, as applicable, is provided, modified to include gene editing at least a portion of the TILs by silencing or suppressing expression of one or more of IL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, ANKRD11, SOCS1, and BCOR.

[0372]

[0392] In another embodiment, the present invention relates to a method for detecting PD-1, CTLA-4, LAG-3, The method of any of the preceding paragraphs, as applicable, is provided, modified to include gene editing at least a portion of the TILs by silencing or suppressing expression of one or more of HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, TIGIT, TOX, ANKRD11, SOCS1, and BCOR.

[0373] D. Overexpression of costimulatory receptors or adhesion molecules

[0393] According to additional embodiments, gene editing of TILs during the TIL expansion method comprises: The expression of one or more immune checkpoint genes is enhanced in at least a portion of the therapeutic TIL population.For example, gene editing can enhance the expression of stimulatory receptors, which means that they are overexpressed compared to the expression of stimulatory receptors that are not genetically modified.Non-limiting examples of immune checkpoint genes that can be enhanced by permanently gene editing the TILs of the present invention include CCR2, CCR4, CCCR5, CCCR6, CCCR7, CCCR8, CCCR9, CCCR10, CCCR11, CCCR12, CCCR13, CCCR14, CCCR15, CCCR16, CCCR17, CCCR18, CCCR19, CCCR20, CCCR21, CCCR22, CCCR23, CCCR24, CCCR25, CCCR26, CCCR27, CCCR28, CCCR29, CCCR30, CCCR31, CCCR32, CCCR33, CCCR40, CCCR41, CCCR51, CCCR52, CCCR53, CCCR61, CCCR72, CCCR83, CCCR94, CCCR105, CCCR116, CCCR126, CCCR137, CCCR143, CCCR154, CCCR165, CCCR176, CCCR187, CCCR198, CCCR19 ...200, CCCR210, CCCR220, CCCR23, CCCR24, CCCR25, CCCR26, CCCR27, CCCR28, CCCR299, CCCR299, CCCR200, CCCR210, CCCR211, CCCR221, CCCR23, CCCR24, CCCR25, CCCR26, CCCR These include certain chemokine receptors and interleukins such as R5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or the NOTCH ligand mDLL1.

[0374] 1. CCR

[0394] For adoptive T cell immunotherapy to be effective, T cells must be targeted to tumors by chemokines. The match between chemokines secreted by tumor cells, chemokines present in the periphery, and chemokine receptors expressed by T cells is crucial for successful trafficking of T cells to the tumor bed.

[0375]

[0395] According to certain embodiments, the gene editing method of the present invention is directed to CCR2, CCR4 These can be used to increase the expression of specific chemokine receptors on TILs, such as one or more of CCR5, CXCR2, CXCR3, and CX3CR1. Overexpression of CCRs helps promote effector function and proliferation of TILs after adoptive transfer.

[0376]

[0396] According to certain embodiments, CCR2, CCR4, CCR5, The expression of one or more of CXCR2, CXCR3, and CX3CR1 is enhanced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by enhancing the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in chemokine receptor genes. For example, CRISPR, TALE, or zinc finger methods can be used to enhance the expression of specific chemokine receptors in TILs.

[0377]

[0397] In one embodiment, the CCR4 and / or CCR5 adhesion molecules are In one embodiment, the CXCR2 adhesion molecule is inserted into the TIL population using gammaretroviral or lentiviral methods as described in Forget, et al., Frontiers Immunology 2017, 8, 908 or Peng, et al., Clin. Cancer Res. 2010, 16, 5458 The vectors are inserted into the TIL population using retroviral or lentiviral methods, the disclosures of which are incorporated herein by reference.

[0378]

[0398] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, the sterile electroporation step comprising: Mediating and carrying out transfers; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step includes delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of PD-1 and optionally LAG-3, and further wherein a CXCR2 adhesion molecule is inserted into the first TIL population, the second TIL population, or the recovered TIL population by gammaretroviral or lentiviral methods.

[0379]

[0399] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system or a zinc finger system to inhibit expression of PD-1 and optionally LAG-3, and further wherein CCR4 and / or CCR5 adhesion molecules are inserted into the first TIL population, the second TIL population or the recovered TIL population by gammaretroviral or lentiviral methods.

[0380]

[0400] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (g) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system or a zinc finger system to inhibit expression of PD-1 and optionally LAG-3, and further wherein an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof is inserted into the first TIL population, the second TIL population or the recovered TIL population by gammaretroviral or lentiviral methods.

[0381] 2. Interleukins

[0401] According to additional embodiments, the gene editing method of the present invention is directed to the expression of IL-2, IL-4, , IL-7, IL-10, IL-15, and IL-21. Certain interleukins have been shown to enhance T cell effector function and mediate tumor control.

[0382]

[0402] According to certain embodiments, IL-2, IL-4, IL-7, The expression of one or more of IL-10, IL-15, and IL-21 is enhanced according to the compositions and methods of the present invention. For example, a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the method described herein (e.g., Process 2A or the method shown in Figures 20 and 21), wherein the method comprises gene editing at least a portion of the TILs by enhancing the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in interleukin genes. For example, CRISPR, TALE, or zinc finger methods can be used to enhance the expression of a particular interleukin in TILs.

[0383]

[0403] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step includes delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of PD-1 and optionally LAG-3, and further includes delivery of an interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, and combinations thereof. is inserted into the first TIL population, the second TIL population, or the harvested TIL population by gammaretroviral or lentiviral methods.

[0384] E. Gene Editing Methods

[0404] As noted above, embodiments of the present invention utilize genetically modified steroids to enhance their therapeutic efficacy. Provided are tumor-infiltrating lymphocytes (TILs) that have been genetically modified via gene editing. Embodiments of the invention encompass gene editing by inserting nucleotides (RNA or DNA) into a TIL population to both promote expression of one or more proteins and inhibit expression of one or more proteins, as well as combinations thereof. Embodiments of the invention also provide methods for expanding TILs into therapeutic populations, the methods comprising genetically editing the TILs. There are several gene editing techniques that can be used to genetically modify TIL populations suitable for use in accordance with the invention.

[0385]

[0405] In some embodiments, the method of genetically modifying a TIL population comprises one or more The method comprises a step of stable integration of a gene for the production of a protein. In one embodiment, the method for genetically modifying a population of TILs comprises a step of retroviral transduction. In one embodiment, the method for genetically modifying a population of TILs comprises a step of lentiviral transduction. Lentiviral transduction systems are known in the art, and are described, for example, in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75;Dull, et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In one embodiment, a 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 one embodiment, a 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, for example, as an mRNA (e.g., an mRNA comprising a cap and polyA tail), so that long-term expression of the transposase does not occur in the transgenic cells. Suitable transposases containing salmonid Tel-like transposases (SB or Sleeping Beauty transposases) such as SB10, SB11, and SB100x are used. Transposon-mediated gene transfer systems and engineered enzymes with improved 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.

[0386]

[0406] In one embodiment, the method of genetically modifying a TIL population comprises the steps of: The method includes a step of stable integration of a gene for protein production or inhibition (e.g., silencing). In one embodiment, the method for genetically modifying a population of TILs includes a 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 / 0227237 A1, the disclosures of each of which are incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Patent Nos. 5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490 (the disclosures of which are incorporated herein by reference). In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, the electroporation method is a pulsed electric field to induce TIL. A pulse electroporation method comprising treating a TIL to alter, manipulate or cause a defined and controlled permanent or transient change in the TIL, the method comprising applying to the TIL a series 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 series of at least three DC electric pulses have 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 of two first sets of at least three pulses differs from a second pulse interval of two second sets of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a defined and controlled permanent or transient change in the TIL, comprising applying to the TIL a series of at least three single-operator-controlled, independently programmed DC electric pulses having an electric field strength of 100 V / cm or greater, wherein at least two of the at least three pulses differ from one another in pulse amplitude. In one embodiment, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a defined and controlled permanent or transient change in the TIL, comprising applying to the TIL a series of at least three single-operator-controlled, independently programmed DC electric pulses having an electric field strength of 100 V / cm or greater, wherein at least two of the at least three pulses differ from one another in pulse width.In one embodiment, the electroporation method is a pulsed electroporation method comprising treating TILs with a pulsed electric field to alter, manipulate or cause defined and controlled permanent or transient changes in the TILs, comprising applying to the TILs a series of at least three single, operator-controlled, independently programmed DC electric pulses having an electric field strength of 100 V / cm or greater, wherein a first pulse interval of two first sets of at least three pulses is different from a second pulse interval of two second sets of at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method, which includes treating TILs with a pulsed electric field to induce pore formation in the TILs, and includes applying a series of at least three DC electric pulses to the TILs, with an electric field strength of 100 V / cm or more, wherein the series of at least three DC electric pulses have one, two, or three of the following characteristics so that the induced pores persist 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 have pulse amplitudes that are different from each other; (2) at least two of the at least three pulses have pulse widths that are different from each other; and (3) a first pulse interval of two first sets of at least three pulses is different from a second pulse interval of two second sets of at least three pulses. In one embodiment, the method for genetically modifying a population of TILs includes 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. Pat. No. 5,593,875. The disclosures of each of which are incorporated herein by reference. In one embodiment, the method for genetically modifying a population of TILs comprises a liposome transfection step. Liposome transfection methods, such as those using a 1:1 (w / w) liposome formulation of the cationic lipids N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphotidylethanolamine (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., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Pat. Nos. 5,279,833; 5,908,635; Same No. 6,056,938; Same No. 6,110,490; Same No. 6,534,484; and and 7,687,070, the disclosures of each of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population comprises a transfection step using the methods described in U.S. Patent Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705, the disclosures of each of which are incorporated herein by reference.

[0387]

[0407] According to one embodiment, the gene editing process involves the use of one or more immune checkpoints. This may involve the use of programmable nucleases that mediate the generation of double- or single-strand breaks in target genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on the recognition of specific DNA sequences within the genome to target the nuclease domain to this location and mediate the generation of double-strand breaks at the target sequence. The double-strand break in DNA then recruits endogenous repair mechanisms to the break site to mediate genome editing via non-homologous end joining (NHEJ) or homology-directed repair (HDR). Thus, repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, suppress, or enhance) the target gene product.

[0388]

[0408] The main nucleases developed to enable site-specific genome editing The major classes of nucleases include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR / Cas9). These nuclease systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, while CRISPR systems, such as Cas9, target specific DNA sequences through short RNA guide molecules that directly base pair with the target DNA and protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

[0389]

[0409] Non-limiting examples of gene editing methods that can be used in accordance with the TIL expansion method of the present invention Typical examples include CRISPR, TALE, and ZFN methods, examples of which are described in more detail below. According to one embodiment, the method of expanding TILs into a therapeutic population can be performed according to any embodiment of the method described herein (e.g., Process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene editing at least a portion of the TILs by one or more of CRISPR, TALE, or ZFN methods to generate TILs that can provide an enhanced therapeutic effect. According to one embodiment, the gene-edited TILs can be evaluated for improved therapeutic effect by comparing them to unmodified TILs in vitro, e.g., by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs.

[0390]

[0410] In some embodiments of the invention, electroporation is performed using a method such as CRISP

[0010] 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 in some embodiments of the present invention is the commercially available MaxCyte STX system. Some other systems that may be suitable for use in the present invention include 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). There are alternative commercially available electroporation instruments. In some embodiments of the invention, the electroporation system forms a closed, sterile system with the remainder of the TIL expansion culture method. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, which forms a closed, sterile system with the remainder of the TIL expansion culture method.

[0391] 1.CRISPR method

[0411] Methods for expanding TILs into therapeutic populations include any of the methods described herein. This method can be carried out according to any of the embodiments (e.g., Process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene editing at least a portion of the TILs 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 silencing or reduction of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of CRISPR methods during the TIL expansion process causes enhancement of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

[0392]

[0412] CRISPR stands for "clustered regularly interspaced short palindromic repeats." " represents a CRISPR system. 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 divided into different types and subtypes. The classification of CRISPR systems is based on the effector Cas protein that can cleave a specific nucleic acid. In Class 1 CRISPR systems, the effector module consists of a multi-protein complex, while Class 2 systems use only a single effector protein. Class 1 CRISPRs include Types I, III, and IV, while Class 2 CRISPRs include Types II, V, and 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), II (exemplified by Cas9), and III (exemplified by Cas10). Type II CRISPR is one of the best-characterized systems.

[0393]

[0413] CRISPR technology has been used to identify naturally occurring genes in bacteria and archaea (the domain of single-celled microorganisms). These organisms have adapted this system from a defense mechanism. They use CRISPR-derived RNA and various Cas proteins, including Cas9, to thwart attacks by viruses and other foreign substances by chopping up and destroying the invader's DNA. CRISPRs are specialized regions of DNA with two distinct characteristics: nucleotide repeats and the presence of spacers. Nucleotide repeats are distributed throughout the CRISPR region, with short segments of foreign DNA (spacers) interspersed between the repeats. In Type II CRISPR / Cas systems, the spacers are integrated into the CRISPR genomic locus, transcribed, and processed into short CRISPR RNAs (crRNAs). These crRNAs anneal to transactivating 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. This allows the CRISPR / Cas system to be retargeted to cleave virtually any DNA sequence by redesigning the crRNA. Thus, according to certain embodiments, Cas9 acts as an RNA-guided DNA endonucleaser that cleaves DNA upon crRNA-tracrRNA recognition. They function as endonucleases. In natural systems, crRNA and tracrRNA 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 spacer of approximately 17–20 nucleotides that defines the genomic target to be modified. Therefore, users can change the genomic target of the Cas protein by altering the target sequence present in the sgRNA. The CRISPR / Cas system can be directly implanted into human cells by co-delivering a plasmid expressing the Cas9 endonuclease and an RNA component (e.g., sgRNA). To reduce targeting limitations, different variants of the Cas protein can be used (e.g., a Cas9 ortholog, such as Cpf1).

[0394]

[0414] According to one embodiment, engineered, programmable, non-naturally occurring A Type II CRISPR-Cas system comprises a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence in a DNA molecule of a TIL. The DNA molecule encodes at least one immune checkpoint molecule, which the TIL expresses. The Cas9 protein cleaves the DNA molecule, thereby altering the expression of the at least one immune checkpoint molecule. The Cas9 protein and guide RNA do not occur together in nature. According to one embodiment, the expression of two or more immune checkpoint molecules is altered. According to one embodiment, the guide RNA comprises a guide sequence fused to a tracr sequence. For example, the guide RNA may comprise a crRNA-tracrRNA or an sgRNA. According to aspects of the present invention, the terms "guide RNA," "single guide RNA," and "synthetic guide RNA" may be used interchangeably and refer to a polynucleotide sequence comprising a guide sequence, which is an approximately 17-20 bp sequence within the guide RNA that specifies a target site.

[0395]

[0415] Cas9 with improved on-target specificity compared to Cas9 Mutants may also be used in accordance with embodiments of the present invention. Such mutants may be referred to as high-fidelity Cas-9. According to one embodiment, a dual-nickase approach may be utilized, in which two nickases targeting opposite DNA strands generate a DSB within the target DNA (often referred to as a double-nick or dual-nickase CRISPR system). For example, this approach may involve mutating one of the two Cas9 nuclease domains, converting Cas9 from a nuclease to a nickase. Non-limiting examples of high-fidelity Cas9 include eSpCas9, SpCas9-HF1, and HypaCas9. Such mutants may reduce or eliminate unwanted changes at non-target DNA sites. See, for example, 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.

[0396]

[0416] Furthermore, according to certain embodiments, improved Cas9 gene delivery into cells, Cas9 scaffolds that improve on-target specificity (such as those disclosed in U.S. Patent Application Publication No. 2016 / 0102324, incorporated herein by reference) can be used. For example, a 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 cleavage of the DNA. These motifs can be combined with each other to create more compact and / or more specific Cas9 scaffolds. Furthermore, motifs can be used to: Split-Cas9 proteins can be generated that are split into two separate RuvC and HNH domains (i.e., reduced or truncated forms of Cas9 proteins or Cas9 mutants containing either the RuvC or HNH domains), which can process target DNA together or separately.

[0397]

[0417] According to certain embodiments, the CRISPR method comprises: This method involves silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas9 nuclease and guide RNA (e.g., crRNA-tracrRNA or sgRNA) containing a sequence of approximately 17-20 nucleotides specific to the target DNA sequence of the TIL. The guide RNA can be delivered as RNA or by transforming 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 target sequence defined by the sgRNA. DSBs can be repaired intracellularly by non-homologous end joining (NHEJ), a mechanism that often results in insertions or deletions (indels) in DNA. Indels often cause frameshifts, for example, by introducing a premature stop codon within the open reading frame (ORF) of the target gene, generating a loss-of-function allele. According to certain embodiments, the result is a loss-of-function mutation in the target immune checkpoint gene.

[0398]

[0418] Instead, DSBs induced by CRISPR / Cas enzymes are expressed in NHE Instead of J, DSBs can be repaired by homology-directed repair (HDR). While NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide changes to large insertions. According to one embodiment, HDR is used to edit immune checkpoint genes by delivering a DNA repair template containing the desired sequence to TILs together with sgRNA and Cas9 or Cas9 nickase. The repair template preferably contains the desired edit as well as additional homologous sequences (often referred to as left and right homology arms) directly upstream and downstream of the target gene.

[0399]

[0419] According to certain embodiments, an enzymatically inactive version of Cas9 (Cas9) is used. sgRNAs (sgRNAs) can target transcription start sites to repress transcription by blocking initiation. Thus, targeted immune checkpoint genes can be repressed without DSBs. dCas9 molecules retain the ability to bind to target DNA based on the sgRNA target sequence. According to one embodiment of the present invention, CRISPR methods involve silencing or reducing expression of one or more immune checkpoint genes by inhibiting or preventing transcription of the target gene. For example, CRISPR methods can involve fusing a transcription repression domain, such as a Krüppel-associated box (KRAB) domain, to an enzymatically inactive version of Cas9, thereby forming, for example, dCas9-KRAB, which targets the transcription start site of an immune checkpoint gene, resulting in inhibition or prevention of gene transcription. Preferably, the repression domain targets a window downstream from the transcription start site, e.g., approximately 500 bp downstream. This approach, which may be referred to as CRISPR interference (CRISPRi), results in robust gene knockdown through reduced transcription of the target RNA.

[0400]

[0420] According to certain embodiments, an enzymatically inactive version of Cas9 (Cas9) is used. CRISPR (s9 or dCas9) can target transcription start sites to activate transcription. This approach can be referred to as CRISPR activation (CRISPRa). According to one embodiment, the CRISPR method involves increasing expression of one or more immune checkpoint genes by activating the transcription of the target gene. According to such an embodiment, the target immune checkpoint gene can be activated without using a DSB. The CRISPR method can include targeting a transcription activation domain to the transcription start site. For example, VP For example, by fusing a transcriptional activator such as VP64 to dCas9, dCas9-VP64 can be formed, targeting the transcription start site of an immune checkpoint gene, resulting in activation of gene transcription. Preferably, the activation domain targets a window upstream, e.g., approximately 50-400 bp downstream, from the transcription start site.

[0401]

[0421] Additional embodiments of the present invention include methods for potently activating target genes in mammalian cells. Activation strategies developed for this purpose may be utilized. Non-limiting examples include dCas9 fused to multiple different activation domains in tandem (e.g., dCas9-VPR), co-expression of epitope-tagged dCas9 with an antibody-activator effector protein (e.g., the SunTag system), or co-expression of dCas9-VP64 with a modified scaffold gRNA and an additional RNA-binding helper activator (e.g., the SAM activator).

[0402]

[0422] According to another embodiment, CRISPR-assisted rational protein engineering (CARP) A CRISPR-mediated genome editing method, referred to as CARPE (CRISPR-mediated genome editing), can be used in accordance with embodiments of the present invention, as disclosed in U.S. Patent No. 9,982,278, incorporated herein by reference. CARPE involves the generation of "donor" and "destination" libraries that directly integrate mutations derived from single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) editing cassettes into the genome. Construction of the donor library involves co-transforming cells with rationally designed editing oligonucleotides using guide RNAs (gRNAs) that hybridize to the target DNA sequence. The editing oligonucleotides are designed to link a PAM deletion or mutation to one or more desired codon mutations in adjacent genes. This allows the entire donor library to be generated in a single transformation. The donor library is then probed by amplification of recombinant chromosomes, such as by PCR, using a synthetic feature from the editing oligonucleotide, i.e., a second PAM deletion or mutation, which is simultaneously incorporated into the 3' end of the gene. This covalently links the codon-targeted mutations that direct the PAM deletion. The donor library is then co-transformed into cells along with the destination gRNA vector to generate a cell population expressing the rationally designed protein library.

[0403]

[0423] According to another embodiment, traceable CRISPR enrichment recombineering (G A method for traceable, precise genome editing using a CRISPR-mediated system, referred to as genome engineering by En-TraCER (En-TraCER), can be used in accordance with embodiments of the present invention, as disclosed in U.S. Patent No. 9,982,278, incorporated herein by reference. The GEN-TraCER method and vector combines an editing cassette and 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 breaks occur and the PAM mutation to be integrated.

[0404]

[0424] Permanently gene-editing TILs via CRISPR methods Non-limiting examples of genes that can be sensed or inhibited include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNF RSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL1 0RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 , GUCY1B3, TOX, ANKRD11, SOCS1 and BCOR.

[0405]

[0425] Enhancement by permanently gene editing TILs via CRISPR methods Non-limiting examples of genes that may be expressed include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

[0406]

[0426] The present invention provides a method for altering the expression of a target gene sequence by CRISPR methods. Examples of systems, methods, and compositions that may be used in accordance with embodiments of the present invention are described in U.S. Patent Nos. 8,697,359; 8,993,233; 8,795,965; 8,771,945; 8,889,356; 8,865,406; 8,999,641; 8,945,839; 8,932,814; 8,871,445; 8,906,616; and 8,895,308, which are incorporated herein by reference. Resources for implementing CRISPR methods, such as plasmids for expressing CRISPR / Cas9 and CRISPR / Cpf1, are available from various sources, including GenScript. Which companies are selling it?

[0407]

[0427] In one embodiment, the genes of a TIL population as described herein The modification can be performed using the CRISPR / Cpf1 system 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.

[0408]

[0428] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step includes delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 or CRISPR / Cpf1 system for regulating expression of at least one protein.

[0409]

[0429] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step involves delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 or CRISPR / Cpf1 system to inhibit expression of PD-1 and LAG-3.

[0410] 2.TALE method

[0430] Methods for expanding TILs into therapeutic populations include any of the methods described herein. This process may be carried out according to any of the embodiments (e.g., Process 2A) or as described in PCT / US Patent Application Publication No. 2017 / 058610, PCT / US Patent Application Publication No. 2018 / 012605, or PCT / US Patent Application Publication No. 2018 / 012633. , wherein the method further comprises gene editing at least a portion of the TILs by the TALE method. According to certain embodiments, the use of the TALE method during the TIL expansion process causes silencing or reduction in expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of the TALE method during the TIL expansion process causes enhancement in expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

[0411]

[0431] TALE stands for TALEN (Transcription Activator-Like Effector Nuclease). TALEs represent "transcription activator-like effector" proteins, including the TALE-like nucleotide sequence (TALE). 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 pathogen Xanthomonas genus. They contain a DNA-binding domain composed of a series of 33-35 amino acid repeat domains, each of which recognizes a single base pair. TALE specificity is determined by two hypervariable amino acids known as repeat variable dinucleotides (RVDs). Modular TALE repeats are joined together to recognize adjacent DNA sequences. Specific RVDs in the DNA-binding domain recognize bases in the target locus and provide the structural features for assembling a predictable DNA-binding domain. The TALE DNA-binding domain is fused to the catalytic domain of type IIS FokI endonuclease to create a targetable TALE nuclease. To induce site-specific mutagenesis, two individual TALEN arms, separated by a 14-20 base pair spacer region, bring FokI monomers into close proximity, allowing them to dimerize and generate a targeted double-stranded break.

[0412]

[0432] Several large-scale systematic studies utilizing various assembly methods have been carried out in TAL These results suggest that E repeats can be combined to recognize virtually any user-defined sequence. Strategies that allow for the rapid assembly of custom TALE arrays include Golden Gate molecular cloning, high-throughput solid-phase assembly, and ligation-independent cloning technologies. Custom-designed TALE arrays are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Furthermore, the ability to recognize the desired target sequence is crucial for the identification of TALEs. Web-based tools are available, such as TAL Effector-Nucleotide Target 2.0, which allow for the design of TALE TAL effector repeat arrays 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 can be found in the US and US Patent Application Publication Nos. 2011 / 0201118 A1; 2013 / 0117869 A1; 2013 / 0315884 A1; 2015 / 0203871 A1 and 2016 / 0120906 A1, the disclosures of which are incorporated herein by reference.

[0413]

[0433] According to one embodiment of the present invention, the TALE method inhibits or amplifies the transcription of a target gene. or silencing or reducing expression of one or more immune checkpoint genes by preventing transcription of the gene. For example, a TALE method can include utilizing a KRAB-TALE, which involves fusing a transcriptional Krüppel-associated box (KRAB) domain to a DNA-binding domain that targets the transcription start site of the gene, resulting in inhibition or prevention of transcription of the gene.

[0414]

[0434] According to another embodiment, the TALE method comprises introducing a mutation into a target gene. For example, the TALE method may involve fusing a nuclease effector domain, such as Fokl, to a TALE DNA binding domain to produce a TALEN. Fokl is active as a dimer, and thus this method involves constructing a pair of TALENs to bind Fokl. This involves positioning an OKL nuclease domain at an adjacent genomic target site, where they introduce a DNA double-strand break. After correct positioning 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 pair (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 deletion, insertion, or replacement of the DNA target site. That is, NHEJ typically results in the introduction of small insertions and deletions at the break site, often inducing a frameshift that knocks out gene function. According to certain embodiments, the TALEN pair targets the majority of the 5' exon of the gene, promoting an early frameshift mutation or a premature stop codon. Gene mutations introduced by TALENs are preferably permanent. Thus, according to one embodiment, the method comprises silencing or reducing expression of an immune checkpoint gene by utilizing a dimerized TALEN to induce a site-specific double-strand break that is repaired via error-prone NHEJ, resulting in one or more mutations in the target immune checkpoint gene.

[0415]

[0435] According to additional embodiments, TALENs are used to induce non-random point mutations, targeted This method is used to introduce genetic changes via HRR, such as deletions or additions of DNA fragments. The introduction of DNA double-strand breaks allows gene editing by homologous recombination in the presence of appropriate donor DNA. According to one embodiment, this method involves 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 DNA.

[0416]

[0436] According to another embodiment, U.S. Patent Application Publication No. 2011 / 0201118 As disclosed, TALENs, which are hybrid proteins derived from FokI and AvrXa7, can be used in accordance with embodiments of the present invention. These TALENs retain the target nucleotide recognition specificity of AvrXa7 and the double-stranded DNA cleavage activity of FokI. Other TALENs with different recognition specificities can be prepared using the same method. 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 Application Publication No. 2013 / 0117869. Selected catalytic domains can be attached to the scaffold to effect DNA processing, which can be designed to ensure that the catalytic domain processes DNA near a single dsDNA target sequence when fused to the core TALE scaffold. Peptide linkers can also be engineered to fuse catalytic domains to the scaffold, creating compact TALENs made from a single polypeptide chain that does not require dimerization to target a specific single dsDNA sequence. The core TALE scaffold can be modified by fusing a catalytic domain, which can be a TAL monomer, to its N-terminus, considering that this catalytic domain can interact with another catalytic domain fused to another TAL monomer, thereby creating a catalytic entity that is more likely to process DNA near the target sequence.See US Patent Application Publication No. 2015 / 0203871.This structure allows for targeting only one DNA strand, which is not an option for traditional TALEN structures.

[0417]

[0437] According to one embodiment of the present invention, conventional RVDs are used to activate gene expression. TALENs can be created that can arbitrarily reduce the number of RVDs. In one embodiment, four RVDs, NI, HD, NN, and NG, are used to target adenine, cytosine, guanine, and thymine, respectively. These conventional RVDs can be used to create TALENs that target, for example, the PD-1 gene. Examples of TALENs that use conventional RVDs include the T3v1 and T1 TALENs disclosed in Gautron et al., Molecular Therapy: Nucleic Acids Dec. 2017, Vol. 9:312-321 (Gautron), which are incorporated herein by reference. T3v1 and T1 TALENs target the PD-L1 binding site at the site of the PD-L1 binding site. The T1 TALEN targets the second exon of the PDCD1 locus, which is located in the PDCD1 gene, and can significantly reduce PD-1 production. In one embodiment, the T1 TALEN does so by using target SEQ ID NO: 127, and the T3v1 TALEN does so by using target SEQ ID NO: 128.

[0418]

[0438] According to another embodiment, TALENs are modified with non-conventional RVDs to improve their activity and specificity for target genes, as disclosed in Gautron. Natural RVDs encompass only a small portion of the potential diversity repertoire of hypervariable amino acid positions. Non-conventional RVDs provide an alternative to natural RVDs and have novel, unique targeting specificity features that can be used to exclude TALENs from targeting off-site targets (sequences in the genome that contain several mismatches compared to the target sequence). Non-conventional RVDs can be used to define arrays, as disclosed in Juillerat, et al., Scientific Reports 5, Article Number 8150 (2015), which is incorporated herein by reference. Non-conventional RVDs can be identified by generating and screening a collection of TALENs containing alternative combinations of amino acids at the two hypervariable amino acid positions. A non-conventional RVD can then be selected that discriminates between the nucleotides present at the mismatched positions, thereby preventing TALEN activity at off-site sequences while allowing for proper processing of the target position. The selected non-conventional RVD can then be used to replace the conventional RVD in the TALEN. Examples of TALENs in which conventional RVDs have been replaced with non-conventional RVDs include the T3v2 and T3v3 PD-1 TALENs produced by Gautron. These TALENs showed improved specificity compared to conventional RVD-based TALENs.

[0419]

[0439] According to additional embodiments, the TALEN silences the expression of two genes. These TALENs can be used to introduce genetic changes that enhance or reduce the specificity of a gene. For example, two separate TALENs can be generated and used together to target two different genes. 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 the analysis of off-target sites and the identification of sites that may be attributable to the use of both TALENs. Based on this information, appropriate conventional and non-conventional RVDs can be selected to design TALENs with improved specificity and activity, even when used together. For example, Gautron used a combination of T3v4 PD-1 and TRAC TALENs to produce potent We disclose generating double knockout CAR T cells that maintain in vitro anti-tumor function.

[0420]

[0440] In one embodiment, TILs may be genetically edited using the method of Gautron or other methods described herein and then expanded by any of the procedures described herein. In some embodiments, the method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) activating a first population of TILs obtained from a tumor resected from a patient for 1 to 5 days using CD3 and CD28 activating beads or antibodies; (b) gene editing at least a portion of the first TIL population using electroporation of a transcription activator-like effector nuclease to obtain a second TIL population; (c) optionally incubating the second population of TILs; (d) performing a first expansion culture by culturing the second TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a third TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and the first expansion culture is performed for about 3 to 14 days to obtain the third TIL population; (e) performing a second expansion culture by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain a fourth TIL population, the fourth TIL population being a therapeutic TIL population; (f) recovering the therapeutic TIL population obtained from step (e); (g) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes; (h) one or more of steps (a) through (g) are carried out in a closed sterile system;

[0421]

[0441] In one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) activating a first population of TILs obtained from a tumor resected from a patient for 1 to 5 days using CD3 and CD28 activating beads or antibodies; (b) gene editing at least a portion of the first TIL population using electroporation of a transcription activator-like effector nuclease in a cytoporation medium to obtain a second TIL population; (c) optionally incubating the second population of TILs; (d) performing a first expansion culture by culturing the second TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a third TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and the first expansion culture is performed for about 6 to 9 days to obtain the third TIL population; (e) performing a second expansion culture by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second expansion culture is performed for about 9 to 11 days to obtain a fourth TIL population, the fourth TIL population being a therapeutic TIL population; (f) recovering the therapeutic TIL population obtained from step (e); (g) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes; (h) one or more of steps (a) through (g) are carried out in a closed sterile system;

[0422]

[0442] In one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) activating a first population of TILs obtained from a tumor resected from a patient for 1 to 5 days using CD3 and CD28 activating beads or antibodies; (b) gene editing at least a portion of the first TIL population using electroporation of a transcription activator-like effector nuclease in a cytoporation medium to obtain a second TIL population; (c) optionally, incubating the second TIL population, wherein the incubation is performed at about 30-40°C with about 5% CO2; (d) performing a first expansion culture by culturing the second TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a third TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and the first expansion culture is performed for about 6 to 9 days to obtain the third TIL population; (e) performing a second expansion culture by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second expansion culture is performed for about 9-10 days to obtain the fourth TIL population. (f) recovering the therapeutic TIL population obtained from step (e); (g) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes; (h) one or more of steps (a) through (g) are carried out in a closed sterile system;

[0423]

[0443] According to another embodiment, TALENs can be specifically designed, such that: Targeting specific selected genes allows for a higher rate of DSB events within target cells. See U.S. Patent Application Publication No. 2013 / 0315884. The use of such rare-cutting endonucleases increases the likelihood of achieving 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 with TALENs to increase mutagenicity and enhance target gene inactivation. The TALENs described in U.S. Patent Application Publication No. 2013 / 0315884 have been successfully used to engineer T cells 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 Application Publication No. 2016 / 0120906. Additionally, TALENs can be used to inactivate genes encoding immunosuppressants and T cell receptor targets, as disclosed in U.S. Patent Application Publication No. 2018 / 0021379, which is incorporated herein by reference. Additionally, 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 Application Publication No. 2019 / 0010514, which is incorporated herein by reference.

[0424]

[0444] Silencing TILs by permanently gene editing them via the TALE method Non-limiting examples of genes that can be targeted or inhibited include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, These include 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, ANKRD11, SOCS1, and BCOR.

[0425]

[0445] Non-limiting examples of TALE nucleases that target the PD-1 gene are listed in the following table: In these examples, the targeted genomic sequence contains two 17 bp long sequences (referred to as half targets and shown in upper case) separated by a 15 base pair (bp) spacer (shown in lower case). Each half target is recognized by a repeat of the half TALE nuclease listed in the table. Thus, according to a particular embodiment, the TALE nuclease according to the present invention recognizes and cleaves a target sequence selected from the group consisting of SEQ ID NO: 127 and SEQ ID NO: 128. TALEN sequences and gene editing methods are also described in Gautron, supra. are.

[0426] [Table 3]

[0427]

[0446] In one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) activating a first population of TILs obtained from a tumor resected from a patient for 1 to 5 days using CD3 and CD28 activating beads or antibodies; (b) gene editing at least a portion of the first TIL population using electroporation of a transcription activator-like effector nuclease that targets PDCD1 in a cytoporation medium to obtain a second TIL population; (c) optionally, incubating the second TIL population, wherein the incubation is performed at about 30-40°C with about 5% CO2; (d) performing a first expansion culture by culturing the second TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a third TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and the first expansion culture is performed for about 6 to 9 days to obtain the third TIL population; (e) performing a second expansion culture by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second expansion culture is performed for about 9 to 11 days to obtain a fourth TIL population, the fourth TIL population being a therapeutic TIL population; (f) recovering the therapeutic TIL population obtained from step (e); (g) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes; (h) one or more of steps (a) through (g) are carried out in a closed sterile system;

[0428]

[0447] In one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) activating a first population of TILs obtained from a tumor resected from a patient for 1 to 5 days using CD3 and CD28 activating beads or antibodies; (b) gene editing at least a portion of the first TIL population using electroporation of a transcription activator-like effector nuclease targeting SEQ ID NO: 128 in cytoporation medium to obtain a second TIL population; (c) optionally, incubating the second TIL population, wherein the incubation is performed at about 30-40°C with about 5% CO2; (d) performing a first expansion culture by culturing the second TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a third TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and the first expansion culture is performed for about 6 to 9 days to obtain the third TIL population; (e) performing a second expansion culture by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second expansion culture is performed for about 9 to 11 days to obtain a fourth TIL population, the fourth TIL population being a therapeutic TIL population; (f) recovering the therapeutic TIL population obtained from step (e); (g) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes; (h) one or more of steps (a) through (g) are carried out in a closed sterile system;

[0429]

[0448] In one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) activating a first population of TILs obtained from a tumor resected from a patient for 1 to 5 days using CD3 and CD28 activating beads or antibodies; (b) gene editing at least a portion of the first TIL population using electroporation of transcription activator-like effector nuclease mRNA according to SEQ ID NO: 135 and SEQ ID NO: 136 in cytoporation medium to obtain a second TIL population; (c) optionally, incubating the second TIL population, wherein the incubation is performed at about 30-40°C with about 5% CO2; (d) performing a first expansion culture by culturing the second TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a third TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, and the first expansion culture is performed for about 6 to 9 days to obtain the third TIL population; (e) performing a second expansion culture by supplementing the cell culture medium of the third TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a fourth TIL population, wherein the second expansion culture is performed for about 9 to 11 days to obtain a fourth TIL population, the fourth TIL population being a therapeutic TIL population; (f) recovering the therapeutic TIL population obtained from step (e); (g) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system. Includes; (h) one or more of steps (a) through (g) are carried out in a closed sterile system;

[0430]

[0449] Enhanced by permanently gene editing TILs via the TALE method Other non-limiting examples of genes to be obtained include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or NOTCH ligand mDLL1.

[0431]

[0450] The present invention provides a method for altering the expression of a target gene sequence by the TALE method. Examples of systems, methods, and compositions that can be used in accordance with the embodiments are described in U.S. Patent No. 8,586,526, which is incorporated herein by reference. These disclosed examples include: Included is the use of non-natural DNA-binding polypeptides having two or more TALE repeat units, including a repeat RVD, an N-cap polypeptide made of residues of a TALE protein, and a C-cap polypeptide made of a fragment of the full-length C-terminal region of a TALE protein.

[0432]

[0451] TALEN design and design strategy, activity evaluation, screening strategy and TA Examples of methods that can be used to efficiently perform LEN-mediated gene integration and inactivation and that may be used in accordance with embodiments of the present invention are described in Valton, et al., Methods, 2014, 69, 151-170, which is incorporated herein by reference.

[0433]

[0452] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step includes delivery of a TALE nuclease system for regulating expression of at least one protein.

[0434]

[0453] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) a first expansion culture by culturing the first TIL population in cell culture medium containing IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days; To carry out; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step includes delivery of a TALE nuclease system to suppress expression of PD-1 and LAG-3.

[0435] 3. Zinc Finger Method

[0454] Methods for expanding TILs into therapeutic populations include any of the methods described herein. This method can be performed according to any of the embodiments (e.g., Process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene editing at least a portion of the TILs by zinc finger or zinc finger nuclease methods. According to certain embodiments, the use of zinc finger methods during the TIL expansion process causes silencing or reduction of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Alternatively, the use of zinc finger methods during the TIL expansion process causes enhancement of expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population.

[0436]

[0455] Each zinc finger contains approximately 30 amino acids in a conserved ββα structure. The zinc finger contains a nucleotide sequence that contains a nucleotide sequence. Several amino acids on the surface of the α-helix typically contact 3 bp of the major groove of DNA with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is the DNA-binding domain, which contains zinc fingers and is found in eukaryotic transcription factors. The second domain is the nuclease domain, which contains the FokI restriction enzyme and is responsible for catalytic cleavage of DNA.

[0437]

[0456] The DNA binding domain of an individual ZFN typically binds 3–6 individual genes. ZFNs contain three-finger repeats, each capable of recognizing 9–18 base pairs. If the zinc finger domains are specific to the target site of interest, a set of three-finger ZFNs recognizing a total of 18 base pairs could theoretically target a single locus in the mammalian genome. One way to generate new zinc finger arrays is to combine smaller zinc finger "modules" with known specificities. 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; see Sangamo Biosciences (Richmond, (St. Louis, MO, USA) in collaboration with Sigma-Aldrich (St. Louis, MO, USA) developed zinc finger structures. A suitable platform (CompoZr®) for this construction has been developed.

[0438]

[0457] By permanently gene editing TILs via zinc finger methodology Non-limiting examples of genes that can be silenced or inhibited include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A. , CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

[0439]

[0458] By permanently gene editing TILs via zinc finger methodology Non-limiting examples of genes that may be enhanced include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

[0440]

[0459] The present invention relates to a method for altering the expression of a target gene sequence by zinc finger methods. Examples of systems, methods, and compositions that may be used in accordance with embodiments of the 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, 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 6,479,626, which are incorporated herein by reference.

[0441]

[0460] The present invention relates to a method for altering the expression of a target gene sequence by zinc finger methods. Other examples of systems, methods, and compositions that may be used in accordance with embodiments of the invention are described in Beane, et al., Mol. Therapy, 2015, 23 1380-1390, the disclosure of which is incorporated herein by reference. will be incorporated into

[0442]

[0461] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) a first expansion culture by culturing the first TIL population in cell culture medium containing IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days; To carry out; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) recovering the therapeutic TIL population obtained from step (g) to provide a recovered TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step includes delivery of a zinc finger nuclease system for regulating expression of at least one protein.

[0443]

[0462] According to one embodiment, tumor infiltrating lymphocytes (TILs) are expanded into a therapeutic TIL population. The method of large-scale cultivation is (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding tumor fragments to a closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, and optionally a 4-1BB agonist antibody, for about 2 to 5 days; (d) adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion culture is performed in a closed vessel that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the therapeutic TIL population obtained from step (g); and wherein the transition from step (g) to step (h) occurs without releasing the system, and the recovered TIL population is a therapeutic TIL population; (i) transferring the recovered TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) Cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step includes delivery of a zinc finger nuclease system to suppress expression of PD-1 and LAG-3.

[0444] IV. TIL Manufacturing Method

[0463] The TIL process known as Process 2A includes some of these characteristics: An example of Process 2A is shown in Figure 1, and some of the advantages of this embodiment of the invention over Process 1C are shown in Figure 2, as well as Figure 14. Process 1C is shown for comparison in Figure 3. Two alternative timelines for TIL treatment based on Process 2A are shown in Figure 4 (high cell number) and Figure 5 (low cell number). An embodiment of Process 2A is shown in Figures 6 and 9. Figures 13 and 14 further provide an exemplary Process 2A compared to exemplary Process 1C.

[0445]

[0464] As discussed herein, the present invention provides a method for the preparation of cryopreserved T cells prior to transplantation into a patient. This may include steps relating to restimulating TILs to increase their metabolic activity, and therefore relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are typically harvested from patient samples and manipulated to expand their numbers prior to transplant into the patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0446]

[0465] In some embodiments, the TILs may be cryopreserved. After thawing, the TILs may be: Before infusion into the patient, it may be restimulated to enhance its metabolism.

[0447]

[0466] In some embodiments, as discussed in detail below and in the Examples and Figures: The first expansion culture (including a process referred to as Pre-REP and shown as step A in FIG. 9 ) is shortened to 3-14 days, and the second expansion culture (including a process referred to as REP and shown as step B in FIG. 9 ) is shortened to 7-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4, 5, and 27 , the first expansion culture (e.g., the expansion culture described as step B in FIG. 9 ) is shortened to 11 days, and the second expansion culture (e.g., the expansion culture described as step D in FIG. 9 ) is shortened to 11 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the combined first and second expansion cultures (e.g., the expansion cultures described as steps B and D in FIG. 9 ) are shortened to 22 days.

[0448]

[0467] The following "step" designations A, B, C, etc. refer to FIG. 9 and are described herein. 9. The order of steps below and in FIG. 9 is exemplary, and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps, is contemplated by the present application and methods disclosed herein.

[0449] A. Step A: Obtain a patient tumor sample

[0468] Generally, TILs are first obtained from patient tumor samples ("primary TILs"). The TILs are then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

[0450]

[0469] Patient tumor samples are generally obtained by surgical extraction using methods known in the art. The tumor sample may be obtained by any means that yields a sample containing a mixture of tumor and TIL cells, such as by radical resection or needle biopsy. Generally, the tumor sample may be from any solid tumor, including primary, invasive, or metastatic tumors. The tumor sample may also be from a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be from any cancer type, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, renal cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, which have been reported to have particularly high levels of TILs.

[0451]

[0470] The term "solid tumor" refers to an abnormal mass of tissue that does not usually contain cysts or fluid 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, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. 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, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The histology of solid tumors comprises interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, in which cancer cells may disperse and provide a supportive microenvironment.

[0452]

[0471] The term "hematologic malignancies" refers to tumors of the blood, bone marrow, lymph nodes, and tissues of the lymphatic system. The term "B-cell hematological malignancies" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0453]

[0472] Once obtained, tumor samples are typically cut into 1 to approximately 8 mm sections using sharp dissection. 3 Small Fragmented into pieces, approximately 2-3 mm 3 are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests are prepared in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg Tumor digests can be produced by incubation in 100 μg / mL gentamicin, 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 enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension at the end of this process contains numerous red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, can be used, the disclosure of which is incorporated herein by reference. Any of the aforementioned methods can be used in any of the embodiments described herein for the methods of expanding TILs or treating cancer.

[0454]

[0473] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" These are called "cell populations."

[0455]

[0474] In some embodiments, fragmentation can be achieved by physical methods, including, for example, cleavage and digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is cleavage. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In one embodiment, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0456]

[0475] In some embodiments, if the tumor is a solid tumor, e.g., step A( After obtaining a tumor sample (as provided in FIG. 9 ), the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for first expansion culture. 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 pieces has a volume of about 1300 mm 3 ~about 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces having a total volume of about 1350 mm 3In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0457]

[0476] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharp dissection. In some embodiments, tumor fragments are about 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 3 In 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 is.

[0458]

[0477] In some embodiments, the TILs are obtained from tumor digests. In embodiments, tumor digests are generated by incubation in enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in enzyme medium, The tumor may be mechanically dissociated for about 1 minute. The solution may then be incubated at 37°C under 5% CO2 for 30 minutes, after which it may again be mechanically disrupted for about 1 minute. After again incubating at 37°C under 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for about 1 minute. In some embodiments, if large tissue debris was present after the third mechanical disruption, the sample was subjected to one or two additional mechanical dissociations, with or without an additional 30-minute incubation at 37°C under 5% CO2. In some embodiments, if the cell suspension at the end of the final incubation contained a large number of red blood cells or dead cells, density gradient separation using Ficoll may be performed to remove such cells.

[0459]

[0478] In some embodiments, the harvested cell suspension prior to the first expansion step The fluid is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0460]

[0479] In some embodiments, the cells are selected from the group consisting of the nuclei described in more detail below and illustrated in FIG. The sample may optionally be frozen after collection and stored frozen before proceeding to expansion as described in step B.

[0461] B. Step B: First Expansion Culture 1.Young TIL

[0480] In some embodiments, the method includes administering to a subject / patient a replication cycle This provides for obtaining young TILs that can expand the number of TILs in a given subject / patient, and thus may provide an additional therapeutic advantage over mature TILs (i.e., TILs that have undergone more rounds of replication before administration to the subject / patient). Characteristics of young TILs have been described in the literature. For example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010), Huang et al., J Immunother, 28(3):258-267 (2005), Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013), Besser et al., J Immunother 32:415-423 (2009), Robbins, et al., J Immunol 2004;173:7125-7130, Shen et al., J Immunother, 30:123-129 (2007), Zhou, et al., J Immunother, 28:53-62 (2005) and Tran, et al., J Immunother, 31:742-751 (2008), all of which and is incorporated herein by reference as set forth herein.

[0462]

[0481] The diverse antigen receptors of T and B lymphocytes are expressed by a limited but numerous gene segments. The TILs are produced by somatic recombination of chromosomes. The V (variable), D (diversity), J (joining), and C (constant) gene segments determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, the TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, the TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 9. In some embodiments, as illustrated in FIG. 13, the TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly harvested TILs and / or TILs prepared using the method designated Process 1C. In some embodiments, the TILs obtained in the first expansion culture exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, expression of T cell receptor (TCR) α and / or β is increased. In some embodiments, expression of T cell receptor (TCR) α is increased. In some embodiments, expression of T cell receptor (TCR) β is increased. In some embodiments, expression of TCRab (i.e., TCR α / β) is increased.

[0463]

[0482] Dissection or digestion of tumor fragments (e.g., as described in step A of Figure 9) The resulting cells are then cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 Bulk TIL cells are obtained. In some embodiments, this primary cell population is cultured for a period of about 11 days, thereby generating a bulk TIL population, generally about 1×10 8 Bulk TIL cells are obtained.

[0464]

[0483] In a preferred embodiment, as described below and herein, an initial ball Expansion of TILs can be performed using a rapid TIL expansion step (e.g., such as that described in step B of FIG. 9, which may include a process referred to as pre-REP), followed by a second expansion (step D, which includes a process referred to as a rapid expansion protocol (REP) step), as described below under step D and herein, followed by optional cryopreservation and then a second step D (which includes a process referred to as a restimulation REP step). TILs obtained by this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0465]

[0484] In an embodiment, TIL cultures are grown in 24-well plates, e.g., Costar 24 wells. Cell culture clusters were initiated using flat-bottom (Corning Incorporated, Corning, NY) When cells were cultured, each well contained 1 × 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 In some embodiments, tumor digest cells or tumor fragments can be seeded. 3 ~10mm 3 is.

[0466]

[0485] In some embodiments, the first expansion culture medium is referred to as " In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The culture is grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 × 10 cells in 10-40 mL of IL-2-containing CM. 6 Live tumor digest cells or 5-30 tumor fragments were loaded. All 24-well plates were incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onwards, half of the medium was replaced every 2–3 days.

[0467]

[0486] After preparation of tumor fragments, the resulting cells (i.e., fragments) are more abundant than tumor and other cells. The primary cells are cultured in serum containing IL-2 under conditions favorable for TIL growth. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2 (or in some cases in the presence of an aAPC cell population as outlined herein). This primary cell population is cultured for several days, generally 10-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion culture contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6 In some embodiments, the IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first expansion culture medium has a final concentration of about 10,000 IU / mL of IL-2, about 9,000 IU In some embodiments, the first expansion culture medium contains about 9,000 IU / mL to about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium contains about 6,000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises 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 one embodiment, the cell culture medium contains 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or about 8000 IU / mL of IL-2.

[0468]

[0487] In some embodiments, the first expansion culture medium contains about 500 IU / mL of I In some embodiments, the first expansion culture medium contains about 500 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 400 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In one preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0469]

[0488] In some embodiments, the first expansion medium contains about 20 IU / mL of IL-1 In some embodiments, the first expansion culture medium contains about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium comprises about 1 IU / mL of IL-21. In one preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[0470]

[0489] In one embodiment, the cell culture medium comprises an OKT-3 antibody. The antibody can be present in the cell culture medium starting on day 0 of REP (i.e., the start of REP) and / or day 0 of the second expansion culture (i.e., the start of the second expansion culture). In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT-3 antibody. In one embodiment, 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 does not contain OKT-3 antibody.

[0471]

[0490] In some embodiments, the first expansion culture medium is referred to as " In some embodiments, this is referred to as "CM." In some embodiments, this is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The culture is grown in a 40 mL volume and 10 cm 2 Gas-permeable flask with a gas-permeable silicone bottom (e.g., G-Rex10; Wilson Wolf In an embodiment initiated in-house (Manufacturing, New Brighton, MN) (Figure 1), each flask was filled with 10-40 x 10 cells in 10-40 mL of IL-2-containing CM. 6 Live tumor digest cells or 5-30 tumor fragments were loaded. Both G-Rex10 and 24-well plates were The cells were incubated at 37°C under 5% CO in a humidified incubator. Five days after the start of the culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onwards, half of the medium was replaced every 2-3 days. In some embodiments, the CM is CM1 described in the Examples (see Example 5). In some embodiments, the first expansion culture is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

[0472]

[0491] In some embodiments, as discussed in the examples and figures, a first expansion The large-scale culture (e.g., including the process described in step B of FIG. 9, which may include what may be referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples, including those discussed in FIGS. 4 and 5 and those discussed in the Examples, including those discussed in step B of FIG. 9, the first expansion culture (e.g., including the process described in step B of FIG. 9, which may include what may be referred to as pre-REP) process is shortened to 7-14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion culture in step B is shortened to 10-14 days. In some embodiments, as discussed in the Examples, including those discussed in FIGS. 4 and 5 and those discussed in step B of FIG. 9, the first expansion culture is shortened to 11 days.

[0473]

[0492] In some embodiments, the first TIL expansion culture is performed on days 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, In some embodiments, the first TIL expansion culture may be continued for 1 day to 14 days. In some embodiments, the first TIL expansion culture may be continued for 2 days to 14 days. In some embodiments, the first TIL expansion culture may be continued for 3 days to 14 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 8 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 11 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 12 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 13 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 14 days. In some embodiments, the first TIL expansion culture may be continued for 1 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 2 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 3 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 8 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 11 days.

[0474]

[0493] In some embodiments, IL-2, IL-7, IL-15 and / or IL In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21, and optional combinations thereof, may be included in the first expansion culture, including in step B process, for example, according to FIG. 9 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination in the first expansion culture. In some embodiments, IL-2, IL-15, and IL-21, and optional combinations thereof, may be included in the step B process, for example, according to FIG. 9 and as described herein. In some embodiments, IL-15 and / or IL-21 are added on day 1. In some embodiments, if the core biopsy is from a pancreatic tumor (e.g., of pancreatic origin), IL-15 and / or IL-21 are added on day 1. In some embodiments, IL-15 is not included. In some embodiments, IL-21 is not included. In some embodiments, neither IL-15 nor IL-21 is included.

[0475]

[0494] In some embodiments, as discussed in the examples and figures, a first expansion The large-scale culture process (including, for example, the process described in step B of FIG. 9, referred to as pre-REP) is shortened to 3 to 14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4 and 5, the first expansion culture in step B is shortened to 7 to 14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4, 5, and 9, the first expansion culture in step B is shortened to 10 to 14 days. In some embodiments, as discussed in the Examples and shown in FIGS. 4, 5, and 9, the first expansion culture in step B is shortened to 11 days.

[0476]

[0495] In some embodiments, the first expansion culture, e.g., step B according to FIG. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, a single bioreactor is used. The actuator is, for example, G-REX-10 or G-REX-100. The bioreactor is a single bioreactor.

[0477]

[0496] In some embodiments, the first expansion culture is 4-1BB, as described below. This is performed using either the agonist antibody and using an additional 4-1BB agonist antibody added to the cell culture medium at the start of expansion culture.

[0478] C. Step C: Transition from the first expansion culture to the second expansion culture

[0497] In some cases, for example, as shown in FIG. 9, The bulk TIL population obtained from the first expansion, including the TIL population obtained from the first expansion, can be immediately cryopreserved using the protocols discussed herein below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which may include an expansion sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, when genetically modified TILs are used therapeutically, the first TIL population (which may also be referred to as the bulk TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) can be subjected to genetic modification for an appropriate treatment before expansion or after the first expansion but before the second expansion.

[0479]

[0498] In some embodiments, from the first expansion culture (e.g., as shown in FIG. 9), The TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 9 ) are stored until phenotyping for selection. In some embodiments, the TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 9 ) are not stored but proceed directly to the second expansion culture. In some embodiments, the TILs obtained from the first expansion culture are not cryopreserved after the first expansion culture and before the second expansion culture. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 3 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 10 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 14 days after fragmentation.

[0480]

[0499] In some embodiments, the transition from the first expansion culture to the second expansion culture comprises: The transition from the first expansion culture to the second expansion culture is performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 1 day to 14 days after fragmentation. In some embodiments, the first TIL expansion culture may be continued for 2 days to 14 days. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 3 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 4 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 5 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 6 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 7 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 8 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is performed 9 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 10 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 11 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 12 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 13 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 1 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 2 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 3 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 4 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 5 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 6 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 7 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 8 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 9 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 10 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 11 days after fragmentation.

[0481]

[0500] In some embodiments, the TILs are cultured after the first expansion and before the second expansion. Rather than being stored, the TILs proceed directly to a second expansion culture (e.g., in some embodiments, no storage occurs during the transition from step B to step D, as shown in FIG. 9). In some embodiments, the transfer occurs in a closed system, as described herein. In some embodiments, the TILs from the first expansion culture are TILs from a second TIL population and proceed directly to a second expansion culture without a transfer period.

[0482]

[0501] In some embodiments, the transition from the first expansion culture to the second expansion culture comprises: For example, step C according to FIG. 9 is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0483] D. Step D: Second Expansion Culture

[0502] In some embodiments, the TIL cell population comprises, for example, as depicted in FIG. After the transition, referred to as Steps A and B and Step C, the numbers increase after harvest and initial bulk processing. This further increase is referred to herein as the second expansion, which may involve an expansion process commonly referred to in the art as rapid expansion (REP and the process shown in Step D of Figure 9). The second expansion can generally be accomplished using culture medium in a gas-permeable vessel containing several components, including feeder cells, a source of cytokines, and an anti-CD3 antibody.

[0484]

[0503] In some embodiments, a second expansion culture of TILs or a second TIL expansion culture Cultivation (which is sometimes referred to as REP, or expansion culture; see step D of Figure 9) The second TIL expansion culture (which may include a culture process) can be performed using any TIL flask or vessel known to those skilled in the art. In some embodiments, the second TIL expansion culture may be continued for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion culture may be continued for about 7 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 8 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 9 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 10 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 11 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 12 to about 14 days. In some embodiments, the second TIL expansion culture may be continued for about 13 to about 14 days. In some embodiments, the second TIL expansion culture may proceed for about 14 days.

[0485]

[0504] In one embodiment, the second expansion culture is performed using the methods of the disclosure (e.g., REP and This process can be performed in a gas-permeable container using a process known as expansion (including the process shown in step D of Figure 9). For example, TILs can be rapidly expanded in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15) using non-specific T cell receptor stimulation. Non-specific T cell receptor stimulation can include, for example, about 30 ng / ml of an anti-CD3 antibody such as OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA), or Examples include UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). The TILs can be expanded to induce further stimulation in vitro by introducing one or more antigens of the cancer, including antigenic portions thereof, such as one or more epitopes of human leukocyte antigen A2 (HLA-A2)-binding peptides, e.g., 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M), optionally expressed from a vector, during the second expansion culture, optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. The TILs can also be rapidly expanded by restimulation with the same one or more antigens of the cancer that were pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second expansion culture. In some embodiments, the second expansion culture occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0486]

[0505] In one embodiment, the cell culture medium further comprises IL-2. In one embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises 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 one embodiment, the cell culture medium contains 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL of IL-2.

[0487]

[0506] In one embodiment, the cell culture medium comprises an OKT3 antibody. The cells were cultured at day 0 of the REP (i.e., the start of the REP) and / or day 0 of the second expansion culture (i.e., The OKT3 antibody may be present in the cell culture medium starting on the first day of the second expansion culture (the initiation day of the second expansion culture). In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of the OKT3 antibody. In one embodiment, 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 OKT3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody.

[0488]

[0507] In some embodiments, IL-2, IL-7, IL-15 and / or IL In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included in the second expansion culture, including during step D of the process, for example, according to Figure 9 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination in the second expansion culture. In some embodiments, a combination of IL-2, IL-15, and IL-21, and any combination thereof, may be included in the second expansion culture, including during step D of the process, for example, according to Figure 9 and as described herein.

[0489]

[0508] In some embodiments, the second expansion culture is cultured in the presence of IL-2, OKT-3, and an anti- The second expansion culture may be performed in a supplemented cell culture medium containing antigen-presenting feeder cells. In some embodiments, the second expansion culture is performed in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion culture occurs with IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0490]

[0509] In some embodiments, the seco...

Claims

1. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from said patient into a plurality of tumor fragments; (b) adding tumor fragments to the closed system, wherein said tumor fragments are from a tumor excised from a patient; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain the second TIL population, the second TIL population being at least 50 times more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; (f) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; and (g) at any point during the method, gene editing at least a portion of the TILs. A method comprising:

2. 10. The method of claim 1, further comprising cryopreserving the infusion bag containing the harvested TIL population in step (f) using a cryopreservation process.

3. 10. The method of claim 1, wherein the cryopreservation process is performed using a 1:1 ratio of the collected TIL population to cryopreservation medium.

4. The method of claim 1 , wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

5. 5. The method of claim 4, wherein the PBMCs are irradiated and allogeneic.

6. 5. The method of claim 4, wherein the PBMCs are added to the cell culture in step (d) on any day from 9 to 14.

7. The method of claim 1 , wherein the antigen-presenting cells are artificial antigen-presenting cells.

8. 10. The method of claim 1, wherein the recovery in step (e) is carried out using a membrane-based cell processing system.

9. 10. The method of claim 1, wherein the harvesting in step (e) is carried out using a LOVO cell processing system.

10. The plurality of fragments includes about 4 to about 50 fragments, each fragment having a length of about 27 mm ３ The method of claim 1 , wherein the volume of the mixture is

11. The plurality of pieces are approximately 1300 mm ３ ~Approx. 1500mm ３ 10. The method of claim 1, wherein the fragments comprise from about 30 to about 60 fragments having a total volume of

12. The plurality of pieces are approximately 1350 mm ３ 10. The method of claim 9, wherein the fragments comprise about 50 fragments having a total volume of 10 ...

13. The method of claim 1 , wherein the plurality of pieces comprises about 50 pieces having a total mass of about 1 gram to about 1.5 grams.

14. 10. The method of claim 1, wherein the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell culture bag.

15. 2. The method of claim 1, wherein the cell culture medium in step (d) further comprises IL-15 and / or IL-21.

16. 16. The method of any one of claims 1 to 15, wherein the IL-2 concentration is from about 10,000 IU / mL to about 5,000 IU / mL.

17. 16. The method of claim 15, wherein the IL-15 concentration is from about 500 IU / mL to about 100 IU / mL.

18. 2. The method of claim 1, wherein the IL-21 concentration is from about 20 IU / mL to about 0.5 IU / mL.

19. The infusion bag in step (f) is a HypoThermosol-containing infusion bag. The method according to claim 1.

20. 4. The method of claim 3, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO).

21. 18. The method of claim 17, wherein the cryopreservation medium comprises 7% to 10% DMSO.

22. 2. The method of claim 1, wherein the first period of time in step (c) and the second period of time in step (e) are each independently performed within a period of 10, 11, or 12 days.

23. 2. The method of claim 1, wherein the first period of time in step (c) and the second period of time in step (e) are each independently performed within a period of 11 days.

24. 10. The method of claim 1, wherein steps (a) through (f) are carried out within a period of about 10 days to about 22 days.

25. 10. The method of claim 1, wherein steps (a) through (f) are carried out within a period of about 20 days to about 22 days.

26. 10. The method of claim 1, wherein steps (a) through (f) are carried out within a period of about 15 days to about 20 days.

27. 10. The method of claim 1, wherein steps (a) through (f) are carried out within a period of about 10 days to about 20 days.

28. 10. The method of claim 1, wherein steps (a) through (f) are carried out within a period of about 10 days to about 15 days.

29. 10. The method of claim 1, wherein steps (a) through (f) are performed in 22 days or less.

30. 10. The method of claim 1, wherein steps (a) through (f) are performed in 20 days or less.

31. 10. The method of claim 1, wherein steps (a) through (f) are carried out in 15 days or less.

32. 10. The method of claim 1, wherein steps (a) through (f) are performed in 10 days or less.

33. 3. The method of claim 2, wherein steps (a) through (f) and cryopreservation are carried out for no more than 22 days.

34. 34. The method of any one of claims 1 to 33, wherein the therapeutic TIL population collected in step (e) comprises sufficient TILs for a therapeutically effective dose of said TILs.

35. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ 35. The method of claim 34, wherein

36. 36. The method of any one of claims 1 to 35, wherein steps (b) through (e) are performed in a single container, and performing steps (b) through (e) in a single container results in an increased yield of TILs per resected tumor compared to performing steps (b) through (e) in multiple containers.

37. The method of any one of claims 1 to 36, wherein the antigen-presenting cells are added to the TILs during a second period in step (d) without releasing the system.

38. 38. The method of any one of claims 1 to 37, wherein the third population of TILs in step (d) provides increased efficacy, increased interferon gamma production, increased polyclonality, increased mean IP-10 and / or increased mean MCP-1 when administered to a subject.

39. 39. The method of any one of claims 1 to 38, wherein the third population of TILs in step (d) provides at least 5-fold or more interferon gamma production when administered to a subject.

40. 40. The method of any one of claims 1 to 39, wherein the third TIL population in step (d) is a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells relative to the second TIL population, and the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression relative to the effector T cells and / or central memory T cells obtained from the second cell population.

41. The effector T cells and / or central memory T cells obtained from the third TIL population are preferably mutated to effector T cells and / or central memory T cells obtained from the second TIL population. The method of any one of claims 1 to 40, wherein the all-memory T cells exhibit increased CD57 expression and decreased CD56 expression.

42. 42. The method of any one of claims 1 to 41, wherein the risk of microbial contamination is reduced compared to open systems.

43. 43. The method of any one of claims 1 to 42, wherein the TILs from step (g) are infused into a patient.

44. 44. The method of any one of claims 1 to 43, wherein the plurality of fragments comprises about 4 fragments.

45. 45. The method of any one of claims 1 to 44, wherein the cell culture medium further comprises a 4-1BB agonist and / or an OX40 agonist during the first expansion culture, the second expansion culture, or both.

46. 46. ​​The method of claim 45, wherein the gene editing is performed after the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

47. 46. ​​The method of claim 45, wherein the gene editing is performed before the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

48. 48. The method of any one of claims 1-47, wherein the gene editing is performed on TILs from one or more of the first population, the second population, and the third population.

49. 48. The method of any one of claims 1 to 47, wherein the gene editing is performed on TILs from the first expansion culture, or TILs from the second expansion culture, or both.

50. 48. The method of any one of claims 1 to 47, wherein the gene editing is performed after the first expansion and before the second expansion.

51. 48. The method of any one of claims 1 to 47, wherein the gene editing is performed before step (c), before step (d), or before step (e).

52. 48. The method of any one of claims 1 to 47, wherein the cell culture medium comprises OKT-3 during the first expansion culture and / or the second expansion culture, and the gene editing is performed before the OKT-3 is introduced into the cell culture medium.

53. 48. The method of any one of claims 1 to 47, wherein the cell culture medium comprises OKT-3 during the first expansion culture and / or the second expansion culture, and the gene editing is performed after the OKT-3 is introduced into the cell culture medium.

54. 48. The method of any one of claims 1-47, wherein the cell culture medium comprises OKT-3 starting on the initiation day of the first expansion culture, and the gene editing is performed after the TILs are exposed to the OKT-3.

55. 55. The method of any one of claims 1-54, wherein the gene editing causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic TIL population.

56. The one or more immune checkpoint genes include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, and CASP 6, 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, ANKRD11, SOCS1 and BCOR.

57. 56. The method of claim 55, wherein the one or more immune checkpoint genes are selected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

58. 55. The method of any one of claims 1-54, wherein the gene editing causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic TIL population, wherein the immune checkpoint genes are selected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

59. 59. The method of any one of claims 1-58, wherein the gene editing comprises the use of a programmable nuclease to mediate the generation of double- or single-stranded breaks in the one or more immune checkpoint genes.

60. 59. The method of any one of claims 1 to 58, wherein the gene editing comprises one or more methods selected from CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

61. 59. The method of any one of claims 1 to 58, wherein the gene editing comprises a CRISPR method.

62. 62. The method of claim 61 , wherein the CRISPR method is a CRISPR / Cas9 method.

63. 59. The method of any one of claims 1 to 58, wherein the gene editing comprises a TALE method.

64. 59. The method of any one of claims 1 to 58, wherein the gene editing comprises zinc finger methods.

65. 1. A method of treating a subject having cancer, comprising administering expanded tumor-infiltrating lymphocytes (TILs), (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from said patient into a plurality of tumor fragments; (b) adding the tumor fragments to the closed system; (c) performing a first expansion by culturing said first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population; wherein the first expansion is carried out in a closed vessel providing a first gas permeable surface area, the first expansion is carried out for about 3 to 14 days to obtain the second TIL population, the second TIL population being at least 50 times more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein the transition from step (d) to step (e) occurs without releasing the system; and (f) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally, cryopreserving the infusion bag containing the recovered TIL population from step (f) using a cryopreservation process; (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the patient; and (i) gene editing at least a portion of the TILs at any time during method steps (a)-(f). The method of claim 1, further comprising administering

66. 66. The method of claim 65, wherein the therapeutic TIL population collected in step (e) comprises sufficient TILs to administer a therapeutically effective dose of the TILs in step (h).

67. The number of TILs sufficient to administer a therapeutically effective dose in step (h) is about 2.3 x 10 １０ ~Approx. 13.7×10 １０ 67. The method of claim 66, wherein

68. 68. The method of claim 67, wherein the antigen-presenting cell (APC) is a PBMC.

69. 69. The method of claim 68, wherein the PBMCs are added to the cell culture in step (d) on any day from 9 to 14.

70. 70. The method of any one of claims 65-69, wherein prior to administering the therapeutically effective dose of TIL cells in step (h), a non-myeloablative lymphodepletion regimen has been administered to the patient.

71. The non-myeloablative lymphodepletion regimen is administered at a dose of 60 mg / m ２ / day for two days, followed by administering cyclophosphamide at a dose of 25 mg / m ２ 71. The method of claim 70, comprising administering fludarabine at a dose of 100 mg / day for 5 days.

72. 72. The method of any one of claims 65-71, further comprising treating said patient with a high-dose IL-2 regimen starting the day after administering said TIL cells to said patient in step (h).

73. 73. The method of claim 72, wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours to a tolerated dose.

74. 74. The method of any one of claims 65 to 73, wherein the third TIL population in step (d) is a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells relative to the second TIL population, and the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression relative to the effector T cells and / or central memory T cells obtained from the second cell population.

75. 75. The method of any one of claims 65-74, wherein the effector T cells and / or central memory T cells in the therapeutic TIL population exhibit increased CD57 expression and decreased CD56 expression relative to effector T cells and / or central memory T cells obtained from the second cell population.

76. 76. The method of any one of claims 65 to 75, 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, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

77. 77. The method of any one of claims 65 to 76, wherein the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer and NSCLC.

78. 78. The method of any one of claims 65 to 77, wherein the cancer is melanoma.

79. 79. The method of any one of claims 65 to 78, wherein the cancer is HNSCC.

80. 80. The method of any one of claims 65 to 79, wherein the cancer is cervical cancer.

81. 81. The method of any one of claims 65 to 80, wherein the cancer is NSCLC.

82. 82. The method of any one of claims 65 to 81, wherein the cell culture medium further comprises a 4-1BB agonist and / or an OX40 agonist during the first expansion culture, the second expansion culture, or both.

83. 83. The method of claim 82, wherein the gene editing is performed after the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

84. 83. The method of claim 82, wherein the gene editing is performed before the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

85. 85. The method of any one of claims 65-84, wherein the gene editing is performed on TILs from one or more of the first population, the second population, and the third population.

86. 85. The method of any one of claims 65 to 84, wherein the gene editing is performed on TILs from the first expansion culture, or TILs from the second expansion culture, or both.

87. 85. The method of any one of claims 65 to 84, wherein the gene editing is performed after the first expansion and before the second expansion.

88. The gene editing is performed before step (c), before step (d), or before step (e). The method according to any one of claims 65 to 84,

89. 89. The method of any one of claims 65-88, wherein the cell culture medium comprises OKT-3 during the first expansion culture and / or the second expansion culture, and the gene editing is performed before the OKT-3 is introduced into the cell culture medium.

90. 89. The method of any one of claims 65-88, wherein the cell culture medium comprises OKT-3 during the first expansion culture and / or the second expansion culture, and the gene editing is performed after the OKT-3 is introduced into the cell culture medium.

91. 89. The method of any one of claims 65-88, wherein the cell culture medium comprises OKT-3 starting on the initiation day of the first expansion culture, and the gene editing is performed after the TILs are exposed to the OKT-3.

92. 92. The method of any one of claims 65-91, wherein the gene editing causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic TIL population.

93. The one or more immune checkpoint genes include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, and CASP 6, 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, ANKRD11, SOCS1 and BCOR.

94. 93. The method of claim 92, wherein the one or more immune checkpoint genes are selected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

95. 92. The method of any one of claims 65-91, wherein the gene editing causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic TIL population, wherein the immune checkpoint genes are selected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

96. 96. The method of any one of claims 65-95, wherein the gene editing comprises the use of a programmable nuclease to mediate the generation of double- or single-stranded breaks in the one or more immune checkpoint genes.

97. The method of any one of claims 65 to 95, wherein the gene editing comprises one or more methods selected from CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

98. 96. The method of claim 65, wherein the gene editing comprises a CRISPR method. How to do it.

99. 99. The method of claim 98, wherein the CRISPR method is a CRISPR / Cas9 method.

100. 96. The method of any one of claims 65 to 95, wherein the gene editing comprises a TALE method.

101. 96. The method of any one of claims 65 to 95, wherein the gene editing comprises zinc finger methods.

102. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) adding processed tumor fragments from a tumor excised from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain the second TIL population, the second TIL population being at least 50 times more numerous than the first TIL population, and the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (b) to step (c) occurs without opening the system; (d) recovering the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without releasing the system; (e) transferring the recovered TIL population from step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; and (f) at any point during the method, gene editing at least a portion of the TILs. A method comprising:

103. 103. The method of claim 102, wherein the therapeutic TIL population collected in step (d) comprises sufficient TILs for a therapeutically effective dose of the TILs.

104. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ The method of claim 103,

105. 105. The method of claim 104, further comprising cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

106. 106. The method of claim 105, wherein the cryopreservation process is performed using a 1:1 ratio of recovered TIL population to cryopreservation medium.

107. 103. The method of claim 102, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

108. 108. The method of claim 107, wherein the PBMCs are irradiated and allogeneic.

109. 109. The method of claim 108, wherein the PBMCs are added to the cell culture in step (c) on any day from 9 to 14.

110. 103. The method of claim 102, wherein the antigen-presenting cell is an artificial antigen-presenting cell.

111. 103. The method of claim 102, wherein said recovering in step (d) is carried out using a LOVO cell processing system.

112. The plurality of fragments includes about 4 to about 50 fragments, each fragment having a length of about 27 mm ３ 63. The method of claim 62, wherein the volume is

113. The plurality of pieces are approximately 1300 mm ３ ~Approx. 1500mm ３ 103. The method of claim 102, comprising about 30 to about 60 fragments having a total volume of

114. The plurality of pieces are approximately 1350 mm ３ 104. The method of claim 103, comprising about 50 fragments having a total volume of

115. 103. The method of claim 102, wherein the plurality of pieces comprises about 50 pieces having a total mass of about 1 gram to about 1.5 grams.

116. 103. The method of claim 102, wherein the plurality of fragments comprises about four fragments.

117. 103. The method of claim 102, wherein the second cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell culture bag.

118. The infusion bag in step (e) is a HypoThermosol-containing infusion bag. The method of claim 102.

119. 103. The method of claim 102, wherein the first period of time in step (b) and the second period of time in step (c) are each independently performed within a period of 10 days, 11 days, or 12 days.

120. 103. The method of claim 102, wherein the first period of time in step (b) and the second period of time in step (c) are each independently performed within a period of 11 days.

121. 103. The method of claim 102, wherein steps (a) through (e) are carried out within a period of about 10 days to about 22 days.

122. 103. The method of claim 102, wherein steps (a) through (e) are carried out within a period of about 10 days to about 20 days.

123. 103. The method of claim 102, wherein steps (a) through (e) are carried out within a period of about 10 days to about 15 days.

124. 103. The method of claim 102, wherein steps (a) through (e) are performed in 22 days or less.

125. 106. The method of claim 105, wherein steps (a) through (e) and cryopreservation are carried out for no more than 22 days.

126. 126. The method of any one of claims 102-125, wherein steps (b)-(e) are performed in a single container, and performing steps (b)-(e) in a single container results in an increased TIL yield per resected tumor compared to performing steps (b)-(e) in multiple containers.

127. The method of any one of claims 102 to 126, wherein the antigen-presenting cells are added to the TILs during the second period in step (c) without releasing the system.

128. The method of any one of claims 102 to 127, wherein the third TIL population in step (d) is a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells relative to the second TIL population, and the effector T cells and / or central memory T cells obtained in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression relative to the effector T cells and / or central memory T cells obtained from the second cell population.

129. 129. The method of any one of claims 102-128, wherein the effector T cells and / or central memory T cells obtained in the therapeutic TIL population exhibit increased CD57 expression and decreased CD56 expression relative to effector T cells and / or central memory T cells obtained from the second cell population.

130. 130. The method of any one of claims 102 to 129, wherein the risk of microbial contamination is reduced compared to open systems.

131. 131. The method of any one of claims 102 to 130, wherein the TILs from step (e) are infused into a patient.

132. 132. The method of any one of claims 102 to 131, wherein the closed vessel comprises a single bioreactor.

133. 133. The method of claim 132, wherein the closed container comprises G-REX-10.

134. 133. The method of claim 132, wherein the closed container comprises G-REX-100.

135. 135. The method of any one of claims 102 to 134, wherein in step (d), the antigen-presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:

1.

136. The cell culture was 2.5 x 10 ９ APCs vs. 100 x 10 ６ The method of claim 135, having a ratio of TILs.

137. 135. The method of any one of claims 102 to 134, wherein in step (c), the antigen-presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:

1.

138. The cell culture was 2.5 x 10 ９ APCs vs. 100 x 10 ６ The method of claim 137, having a ratio of TILs.

139. The cell culture medium may be 4-1B during the first expansion culture, the second expansion culture, or both.

139. The method of any one of claims 102 to 138, further comprising a B agonist and / or an OX40 agonist.

140. 140. The method of claim 139, wherein the gene editing is performed after the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

141. 140. The method of claim 139, wherein the gene editing is performed before the 4-1BB agonist and / or the OX40 agonist is introduced into the cell culture medium.

142. 142. The method of any one of claims 102-141, wherein the gene editing is performed on TILs from one or more of the first population, the second population, and the third population.

143. 142. The method of any one of claims 102-141, wherein the gene editing is performed on TILs from the first expansion culture, or TILs from the second expansion culture, or both.

144. 142. The method of any one of claims 102 to 141, wherein the gene editing is performed after the first expansion and before the second expansion.

145. 142. The method of any one of claims 102-141, wherein the gene editing is performed before step (b), before step (c), or before step (d).

146. 146. The method of any one of claims 102-145, wherein the cell culture medium comprises OKT-3 during the first expansion culture and / or the second expansion culture, and the gene editing is performed before the OKT-3 is introduced into the cell culture medium.

147. 146. The method of any one of claims 102-145, wherein the cell culture medium comprises OKT-3 during the first expansion culture and / or the second expansion culture, and the gene editing is performed after the OKT-3 is introduced into the cell culture medium.

148. 146. The method of any one of claims 102-145, wherein the cell culture medium comprises OKT-3 starting on the initiation day of the first expansion culture, and the gene editing is performed after the TILs are exposed to the OKT-3.

149. 149. The method of any one of claims 102-148, wherein the gene editing causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic TIL population.

150. The one or more immune checkpoint genes include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, and CASP 6, 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, ANKRD11, SOCS1 and BCOR.

151. 150. The method of claim 149, wherein the one or more immune checkpoint genes are selected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

152. 149. The method of any one of claims 102-148, wherein the gene editing causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic TIL population, wherein the immune checkpoint genes are selected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

153. 153. The method of any one of claims 102-152, wherein the gene editing comprises the use of a programmable nuclease to mediate the generation of double- or single-stranded breaks in the one or more immune checkpoint genes.

154. The method of any one of claims 102 to 152, wherein the gene editing comprises one or more methods selected from CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

155. 153. The method of any one of claims 102 to 152, wherein the gene editing comprises a CRISPR method.

156. 156. The method of claim 155, wherein the CRISPR method is a CRISPR / Cas9 method.

157. The method of any one of claims 102 to 152, wherein the gene editing comprises a TALE method.

158. 153. The method of any one of claims 102 to 152, wherein the gene editing comprises zinc finger methods.

159. 1. An expanded cultured TIL population for use in treating a subject with cancer, comprising: (a) obtaining a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from said subject into a plurality of tumor fragments; (b) adding the tumor fragments to the closed system; (c) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain the second TIL population, the second TIL population being at least 50 times more numerous than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), wherein The transition from step (d) to step (e) occurs without opening the system; (f) transferring the recovered TIL population from step (e) to an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system; (g) optionally, cryopreserving the infusion bag containing the recovered TIL population from step (f) using a cryopreservation process; and (h) at any point during the method, gene editing at least a portion of the TILs. A third TIL population, an expanded TIL population, obtainable by a method comprising:

160. 160. A TIL population for use in treating a subject with cancer according to claim 159, wherein the method further comprises one or more of the features of any one of claims 1 to 158.

161. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein the cell culture medium further comprises a 4-1BB agonist and / or an OX40 agonist during the first expansion culture, the second expansion culture, or both.

162. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein the gene editing is performed on TILs from one or more of the first population, the second population, and the third population.

163. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein the gene editing is performed on TILs from the first expansion culture or TILs from the second expansion culture, or both.

164. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein the gene editing is performed after the first expansion and before the second expansion.

165. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein the gene editing is performed before step (c), before step (d), or before step (e).

166. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein the gene editing causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the expanded TIL population.

167. The one or more immune checkpoint genes include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FAD 167. A TIL population for use in treating a subject with cancer according to claim 166, wherein the TIL population is selected from the group comprising: D, 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, ANKRD11, SOCS1 and BCOR.

168. 167. The TIL population for use in treating a subject with cancer of claim 166, wherein the one or more immune checkpoint genes are selected from the group including PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

169. 167. The TIL population for use in treating a subject with cancer of claim 166, wherein said gene editing causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of said therapeutic TIL population, wherein said immune checkpoint genes are selected from the group including CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD) and / or NOTCH ligand mDLL1.

170. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein said gene editing comprises the use of a programmable nuclease to mediate the creation of double- or single-stranded breaks in said one or more immune checkpoint genes.

171. 160. The TIL population for use in treating a subject with cancer of claim 159, wherein the gene editing comprises one or more methods selected from CRISPR methods, TALE methods, zinc finger methods, and combinations thereof.

172. 160. A cryopreservation composition comprising a TIL population, a cryoprotective medium comprising DMSO, and an electrolyte solution for use in treating a subject with cancer as described in claim 159.

173. 173. The cryopreserved composition of claim 172, further comprising one or more stabilizers and one or more lymphocyte growth factors.

174. 174. The cryopreserved composition of claim 173, wherein the one or more stabilizers comprise human serum albumin (HSA) and the one or more lymphocyte growth factors comprise IL-2.

175. 175. The cryopreservation composition of claim 174, wherein the cryoprotective medium comprising DMSO and the electrolyte solution are present in a ratio of about 1.1:1 to about 1:1.

1.

176. Approximately 1×10 ６ ~Approx. 9×10 １４ 175. The cryopreservation composition of claim 174, comprising: an amount of the TIL population; an amount of the cryoprotective medium comprising DMSO in an amount of about 30 mL to about 70 mL; an amount of the electrolyte solution in an amount of about 30 mL to about 70 mL; an amount of HSA in an amount of about 0.1 g to about 1.0 g; and an amount of IL-2 in an amount of about 0.001 mg to about 0.005 mg.

177. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from said patient into a plurality of tumor fragments; (b) adding the tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 2 to 5 days; (d) optionally adding OKT-3 to generate a second TIL population, wherein said first expansion is performed in a closed container providing a first gas permeable surface area, said first expansion is performed for about 1 to 3 days to obtain a second TIL population, said second TIL population being at least 50-fold more numerous than said first TIL population, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on said second TIL population, wherein said sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs). conducting a second expansion culture to generate a third TIL population by: (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), TOX, ANKRD11, SOCS1, BCOR, and combinations thereof.

178. 178. The method of claim 177, comprising performing the first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, OKT-3, and a 4-1BB agonist antibody, wherein the OKT-3 and the 4-1BB agonist antibody are optionally present in the cell culture medium starting on day 0 or day 1.

179. 179. The method of claim 177 or 178, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof.

180. 179. The method of claim 177 or 178, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of PD-1.

181. 179. The method of claim 177 or 178, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of LAG-3.

182. 179. The method of claim 177 or 178, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of TIM-3.

183. 179. The method of claim 177 or 178, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of CISH.

184. The electroporation step involves the use of cluster markers to inhibit the expression of CBLB.

179. The method of claim 177 or 178, comprising delivery of a Critical Interspaced Short Palindromic Repeats (CRISPR) system, a Transcription Activator-Like Effector (TALE) system or a Zinc Finger system.

185. 185. The method of any one of Claims 177-184, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system.

186. 185. The method of any one of claims 177-184, wherein the electroporation step comprises delivery of a transcription activator-like effector (TALE) system.

187. 185. The method of any one of claims 177 to 184, wherein the electroporation step comprises delivery of a zinc finger system.

188. 188. The method of any one of claims 177-187, wherein the dimethyl sulfoxide-based cryopreservation medium comprises DMSO, an electrolyte solution, optionally HSA, and optionally IL-2.

189. 1. A method of treating a subject having cancer, comprising: The method comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from said patient into a plurality of tumor fragments; (b) adding the tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 2 to 5 days; (d) optionally, adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium; and (k) administering a therapeutically effective dose of the recovered population of TILs from the infusion bag to the patient. Giving wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), TOX, ANKRD11, SOCS1, BCOR, and combinations thereof.

190. 190. The method of claim 189, comprising performing the first expansion by culturing the first TIL population in cell culture medium comprising IL-2, OKT-3, and a 4-1BB agonist antibody, wherein the OKT-3 and the 4-1BB agonist antibody are optionally present in the cell culture medium starting on day 0 or day 1.

191. 191. The method of claim 189 or 190, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof.

192. 191. The method of claim 189 or 190, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of PD-1.

193. 191. The method of claim 189 or 190, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of LAG-3.

194. 191. The method of claim 189 or 190, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of TIM-3.

195. 191. The method of claim 189 or 190, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of CISH.

196. 191. The method of claim 189 or 190, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of CBLB.

197. 197. The method of any one of claims 189-196, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system.

198. The electroporation step involves the addition of transcription activator-like effectors (TALEs) 197. The method of any one of claims 189 to 196, comprising delivery of a system.

199. 197. The method of any one of claims 189 to 196, wherein the electroporation step comprises delivery of a zinc finger system.

200. 197. The method of any one of claims 189-196, wherein the dimethyl sulfoxide-based cryopreservation medium comprises DMSO, an electrolyte solution, optionally HSA, and optionally IL-2.

201. 1. An expanded cultured TIL population for use in treating a subject with cancer, comprising: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from said patient into a plurality of tumor fragments; (b) adding the tumor fragments to the closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 2 to 5 days; (d) optionally, adding OKT-3 to generate a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, the first expansion is performed for about 1-3 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a sterile electroporation step on the second population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the second population of TILs to rest for about 1 day; (g) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 11 days to obtain the third TIL population, the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) cryopreserving the recovered TIL population using a dimethyl sulfoxide-based cryopreservation medium. wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), TOX, ANKRD11, SOCS1, BCOR, and combinations thereof.

202. performing the first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, OKT-3, and a 4-1BB agonist antibody, wherein the OKT-3 and the 4-1BB agonist antibody are optionally administered on day 0 or day 1; 202. The expanded TIL population of claim 201, wherein the TIL population is initially present in said cell culture medium.

203. 203. The expanded TIL population of claims 201 or 202, wherein said electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof.

204. 203. The expanded TIL population of claim 201 or 202, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of PD-1.

205. 203. The expanded TIL population of claim 201 or 202, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of LAG-3.

206. The expanded TIL population of claims 201 or 202, wherein the electroporation step includes delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of TIM-3.

207. 203. The expanded TIL population of claim 201 or 202, wherein the electroporation step includes delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of CISH.

208. 203. The expanded TIL population of claim 201 or 202, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system to inhibit expression of CBLB.

209. 209. The expanded cultured TIL population of any one of claims 201-208, wherein the electroporation step comprises delivery of a clustered regularly interspaced short palindromic repeats (CRISPR) system.

210. 209. The expanded TIL population of any one of claims 201-208, wherein the electroporation step comprises delivery of a transcription activator-like effector (TALE) system.

211. 209. The expanded cultured TIL population of any one of claims 201-208, wherein the electroporation step comprises delivery of a zinc finger system.

212. 212. The expanded TIL population of any one of claims 201-211, wherein the dimethyl sulfoxide-based cryopreservation medium comprises DMSO, an electrolyte solution, optionally HSA, and optionally IL-2.

213. 212. The expanded cultured TIL population of any one of claims 201 to 211, wherein the electroporation step comprises a pulsed electroporation step.

214. 59. The method of any one of claims 1 to 58, wherein the gene editing comprises a CRISPR method, and the CRISPR method comprises the use of high-fidelity Cas9.

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