Process for generating TIL products with PD-1 TALEN knockdown

Abstract

The present invention provides an improved method for expanding TILs and producing therapeutic TIL populations, including a method for gene editing at least a portion of TILs to enhance their therapeutic efficacy. This method provides improved efficacy, improved phenotype, and increased metabolic health of TILs in a shorter period of time, while allowing for reduced microbial contamination and reduced costs. Such TILs find use in therapeutic treatment regimens.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 242,373, filed September 9, 2021, 63 / 287,670, filed December 9, 2021, 63 / 322,190, filed March 21, 2022, 63 / 354,605, filed June 22, 2022, and 63 / 394,248, filed August 1, 2022, the disclosures of which are incorporated herein in their entireties. [Background technology]

[0002] The treatment of bulky, refractory cancers using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) offers a powerful approach to treating patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. T cells predominate among TILs, and IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become the preferred method of 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. Many approaches to improving response to TIL therapy in melanoma and extending TIL therapy to other tumor types have met with limited success, and the field remains challenging. Goff, et al., J. Clin. Oncol. 2016, 34, 2389-97; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Rosenberg, et al., Clin. Cancer Res. 2011, 17, 4550-57. Combination studies with single immune checkpoint inhibitors have also been described, but further studies are ongoing and additional treatment options are needed (Kverneland, et al., Oncotarget, 2020, 11(22), 2092-2105).

[0003] Furthermore, current TIL production and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, thus significantly limiting the ability to treat patients who are refractory to checkpoint inhibitor therapy. There is an urgent need to provide TIL production processes and therapies based on such processes that are suitable for use in treating patients who have few or no remaining viable treatment options. The present invention fills this need by providing an abbreviated production process for use in generating TILs.

[0004] The present invention provides improved and / or shortened processes and methods for expanding TILs and producing therapeutic TIL populations, including methods for gene editing at least a portion of the therapeutic TIL population to enhance their therapeutic efficacy. Summary of the Invention

[0005] Provided herein are methods for expanding TILs and producing therapeutic TIL populations, including methods for gene editing at least a portion of the TILs to enhance their therapeutic efficacy.

[0006] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population for 1 to 7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) culturing the fourth TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0007] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) culturing a first population of TILs obtained and / or received from tumor tissue resected from a subject or patient in a first cell culture medium containing IL-2 for about 3-9 days to produce a second population of TILs; (b) activating the second TIL population for 1 to 7 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to produce a third TIL population; (c) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (d) culturing the fourth TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0008] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 3 to 8 days; (c) activating the second TIL population for 1 to 6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) performing a second rapid expansion of the fourth TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3, and APC, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0009] In some embodiments, provided herein are methods of expanding tumor-infiltrating lymphocytes into a therapeutic TIL population, the methods comprising: (a) obtaining and / or receiving a first population of TILs from a tumor tissue sample made by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject; (b) adding the tumor tissue to a closed system and performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 9 days to obtain the second TIL population; (c) activating the second TIL population using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, for 1 to 7 days to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; (e) performing a second expansion of the fourth TIL population by culturing it in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a fifth TIL population, wherein the second expansion is performed for about 5-15 days to obtain the fifth TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, wherein the fifth TIL population is a therapeutic TIL population; (f) harvesting the therapeutic TIL population obtained from step (e), wherein each of steps (b) through (f) is performed in a closed sterile system, and the transition from step (b) to step (c), from step (c) to step (d), from step (d) to step (e), and / or the transition from step (e) to step (f) occurs without opening the system.

[0010] In some embodiments, the method comprises: The method further comprises digesting the tumor tissue in an enzymatic medium to produce a tumor digest.

[0011] In some embodiments, the enzyme medium comprises DNase.

[0012] In some embodiments, the enzyme medium comprises collagenase.

[0013] In some embodiments, the enzyme medium comprises a neutral protease.

[0014] In some embodiments, the enzyme medium comprises hyaluronidase.

[0015] In some embodiments, the step of culturing or rapidly expanding the fourth TIL population is carried out by culturing the fourth TIL population in a second cell culture medium for a first period of about 1 to 7 days, at the end of the first period, splitting the fourth TIL population into multiple subcultures, each of the subcultures being cultured in a third cell culture medium containing IL-2 for a second period of about 3 to 7 days, and at the end of the second period, the subcultures being combined to provide an expanded number of TILs or a therapeutic TIL population.

[0016] In some embodiments, the first culture period is about 5 days.

[0017] In some embodiments, the second culture period is about 4 days.

[0018] In some embodiments, the second culture period is about 5 days.

[0019] In some embodiments, the step of activating the second TIL population is carried out using anti-CD3 agonist beads or antibodies.

[0020] In some embodiments, activating the second TIL population is performed using OKT-3.

[0021] In some embodiments, the step of activating the second TIL population is performed using OKT-3 at 300 ng / mL.

[0022] In some embodiments, the step of activating the second TIL population is performed using anti-CD3 agonist and anti-CD28 agonist beads or antibodies.

[0023] In some embodiments, activating the second TIL population is performed using TransAct.

[0024] In some embodiments, the step of activating the second TIL population is performed using TransAct at a dilution of 1:10, 1:17.5, or 1:100.

[0025] In some embodiments, the step of activating the second population of TILs is carried out for about 2 days.

[0026] In some embodiments, the step of activating the second population of TILs is carried out for about 3 days.

[0027] In some embodiments, the step of activating the second population of TILs is carried out for about 4 days.

[0028] In some embodiments, the step of activating the second population of TILs is carried out for about 5 days.

[0029] In some embodiments, the step of culturing the first population of TILs is carried out for about 3 days.

[0030] In some embodiments, the step of culturing the first population of TILs is carried out for about 5 days.

[0031] In some embodiments, the step of culturing the first population of TILs is carried out for about 7 days.

[0032] In some embodiments, the step of culturing the fourth population of TILs is carried out for about 8 days.

[0033] In some embodiments, the step of culturing the fourth population of TILs is carried out for about 9 days.

[0034] In some embodiments, the step of culturing the fourth TIL population is carried out for about 8-9 days.

[0035] In some embodiments, the step of culturing the fourth population of TILs is carried out for about 10 days.

[0036] In some embodiments, the step of culturing the fourth TIL population is carried out for about 8-10 days.

[0037] In some embodiments, all steps are completed within a period of about 22 days.

[0038] In some embodiments, all steps are completed within a period of about 19-22 days.

[0039] In some embodiments, all steps are completed within a period of about 19-20 days.

[0040] In some embodiments, all steps are completed within a period of about 20-22 days.

[0041] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3 to 9 days to produce a second TIL population; (c) gene editing at least a portion of the second TIL population to produce a third TIL population; (d) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0042] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) culturing a first population of TILs obtained and / or received from tumor tissue resected from a subject or patient in a first cell culture medium comprising IL-2 and OKT-3 for about 3-9 days to produce a second population of TILs; (b) gene editing at least a portion of the second TIL population to produce a third TIL population; (c) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0043] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 3 to 8 days; (c) gene editing at least a portion of the second TIL population to produce a third TIL population; (d) performing a rapid second expansion of the third TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3, and APC, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0044] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) performing an initial expansion (or first expansion by priming) of a first TIL population obtained and / or received from tumor tissue resected from a subject or patient in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 3 to 8 days; (b) gene editing at least a portion of the second TIL population to produce a third TIL population; (c) performing a rapid second expansion of the third TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3, and APC, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0045] In some embodiments, the method comprises: The method further comprises digesting the tumor tissue in an enzymatic medium to produce a tumor digest.

[0046] In some embodiments, the enzyme medium comprises DNase.

[0047] In some embodiments, the enzyme medium comprises collagenase.

[0048] In some embodiments, the enzyme medium comprises a neutral protease.

[0049] In some embodiments, the enzyme medium comprises hyaluronidase.

[0050] In some embodiments, provided herein are methods for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) culturing a first population of TILs obtained by digesting tumor tissue excised from a subject or patient in an enzymatic medium to produce a tumor digest in a first cell culture medium containing IL-2 and OKT-3 for about 3 to 9 days to produce a second population of TILs; (b) gene editing at least a portion of the second TIL population to produce a third TIL population; (c) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0051] In some embodiments, the step of culturing or initially expanding the first TIL population comprises culturing the first TIL population in a first cell culture medium comprising IL-2 for about 3 days, followed by culturing the first TIL population in a cell culture medium comprising IL-2 and OKT-3 for 2-6 days.

[0052] In some embodiments, the step of culturing or rapidly expanding the third TIL population is carried out by culturing the third TIL population in a second cell culture medium for a first period of about 1-7 days, at the end of the first period, dividing the third TIL population into multiple subcultures, each of which is cultured in a third cell culture medium containing IL-2 for a second period of about 3-7 days, and at the end of the second period, combining the subcultures to provide an expanded number of TILs or a therapeutic TIL population.

[0053] In some embodiments, the first culture period is about 5 days.

[0054] In some embodiments, the second culture period is about 4 days.

[0055] In some embodiments, the second culture period is about 5 days.

[0056] In some embodiments, the step of culturing the first population of TILs is carried out for about 3 days.

[0057] In some embodiments, the step of culturing the first population of TILs is carried out for about 5 days.

[0058] In some embodiments, the step of culturing the first population of TILs is carried out for about 7 days.

[0059] In some embodiments, the step of culturing the third population of TILs is carried out for about 8 days.

[0060] In some embodiments, the step of culturing the third population of TILs is carried out for about 9 days.

[0061] In some embodiments, the step of culturing the third TIL population is carried out for about 8 to 9 days.

[0062] In some embodiments, the step of culturing the third population of TILs is carried out for about 10 days.

[0063] In some embodiments, the step of culturing the third TIL population is carried out for about 8-10 days.

[0064] In some embodiments, all steps are completed within a period of about 22 days.

[0065] In some embodiments, all steps are completed within a period of about 20 days.

[0066] In some embodiments, all steps are completed within a period of about 22 days.

[0067] In some embodiments, all steps are completed within a period of about 19-22 days.

[0068] In some embodiments, all steps are completed within a period of about 19-20 days.

[0069] In some embodiments, all steps are completed within a period of about 20-22 days.

[0070] In some embodiments, all steps are completed within a period of about 16-18 days.

[0071] In some embodiments, the culturing or initial expansion step of the first TIL population in the first culture medium further comprises anti-CD3 and anti-CD28 beads or antibodies.

[0072] In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct.

[0073] In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies include TransAct at a dilution of 1:10, 1:17.5, or 1:100.

[0074] In some embodiments, the first culture medium comprises OKT-3 at 300 ng / mL.

[0075] In some embodiments, the step of culturing or initially expanding the first TIL population comprises culturing the first TIL population in a first cell culture medium comprising IL-2 and anti-CD3 and anti-CD28 beads or antibodies for about 3 days, followed by culturing the first TIL population in a cell culture medium comprising IL-2 and OKT-3 for 2-4 days.

[0076] In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies comprise TransAct.

[0077] In some embodiments, the anti-CD3 and anti-CD28 beads or antibodies include TransAct at a dilution of 1:10, 1:17.5, or 1:100.

[0078] In some embodiments, the first culture medium comprises OKT-3 at 300 ng / mL.

[0079] In some embodiments, the expanded number of TILs comprises a therapeutic TIL population.

[0080] In some embodiments, the step of gene editing at least a portion of the second or third TIL population comprises performing a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

[0081] In some embodiments, the step of gene editing at least a portion of the second or third TIL population comprises performing a sterile electroporation step on the second or third TIL population, wherein the sterile electroporation step mediates the transfer of at least two gene editors.

[0082] In some embodiments, the electroporation step consists of a single electroporation event that mediates the transfer of at least two gene editors.

[0083] In some embodiments, in the electroporation step, each of the at least two gene editors is individually introduced by an electroporation event, independent of the introduction of any other gene editor.

[0084] In some embodiments, the electroporation step further comprises a rest period after each electroporation event.

[0085] In some embodiments, the electroporation step comprises a first electroporation event mediating the transfer of a first gene editor to modulate expression of a first protein, a first resting period, a second electroporation event mediating the transfer of a second gene editor to modulate expression of a second protein, and a second resting period, wherein the first and second resting periods are the same or different.

[0086] In some embodiments, the first and second resting periods comprise incubating the third or fourth TIL population in a second cell culture medium comprising IL-2 and / or IL-15.

[0087] In some embodiments, the first and second resting periods comprise incubating the third or fourth TIL population in a second cell culture medium containing IL-2 at 300 IU / mL, 1000 IU / mL, or 6000 IU / mL.

[0088] In some embodiments, the first and second resting periods comprise incubating the third or fourth TIL population in a second cell culture medium comprising IL-15 at 15 ng / mL.

[0089] In some embodiments, the first and second resting periods comprise incubating the third or fourth TIL population at about 30-40° C. and about 5% CO 2 .

[0090] In some embodiments, the first and second resting periods comprise incubating the third or fourth TIL population at about 25, 28, 30, 32, 35, or 37° C. and about 5% CO 2 .

[0091] In some embodiments, the first and second resting periods are independently from about 10 hours to 5 days.

[0092] In some embodiments, the first and second resting periods are independently from about 10 hours to 3 days.

[0093] In some embodiments, the first resting period is about 1 to 3 days.

[0094] In some embodiments, the first resting period is about 3 days.

[0095] In some embodiments, the second resting period is from about 10 hours to 1 day.

[0096] In some embodiments, the second resting period is about 12 to 24 hours.

[0097] In some embodiments, the second resting period is from about 15 hours to about 18 hours.

[0098] In some embodiments, the second resting period comprises incubating the third or fourth TIL population in cell culture medium containing IL-2 at about 30° C. for about 15 to 23 hours.

[0099] In some embodiments, the second resting period comprises incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about 1 hour, followed by incubation at about 30°C for about 15 to 23 hours.

[0100] In some embodiments, the second resting period comprises incubating the third or fourth TIL population in cell culture medium containing IL-2 at 37°C for about 1 hour, followed by about 15 to 22 hours at about 30°C.

[0101] In some embodiments, the first resting period is about 3 days and the second resting period is about 10-16 hours.

[0102] In some embodiments, the second or third TIL population is washed in a cytoporation buffer prior to the electroporation step.

[0103] In some embodiments, the at least one gene editor is a TALE nuclease system for regulating expression of at least one protein.

[0104] In some embodiments, at least one gene editor comprises a TALE nuclease system that regulates expression of PD-1.

[0105] In some embodiments, at least one gene editor comprises a TALE nuclease system that regulates expression of CTLA-4.

[0106] In some embodiments, at least one gene editor comprises a TALE nuclease system that regulates expression of LAG-3.

[0107] In some embodiments, at least one gene editor comprises a TALE nuclease system that regulates expression of CISH.

[0108] In some embodiments, at least one gene editor comprises a TALE nuclease system that regulates expression of CBL-B.

[0109] In some embodiments, at least one gene editor comprises a TALE nuclease system that regulates expression of TIGIT.

[0110] In some embodiments, the at least two gene editors comprise a first gene editor comprising a first TALE-nuclease system for regulating expression of a first protein, and a second gene editor comprising a second TALE-nuclease system for regulating expression of a second protein.

[0111] In some embodiments, the first and second TALE-nuclease systems regulate the expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT and / or CBL-B.

[0112] In some embodiments, the first and second TALE-nuclease systems regulate the expression of PD-1 and CTLA-4.

[0113] In some embodiments, the first and second TALE-nuclease systems regulate the expression of PD-1 and LAG-3.

[0114] In some embodiments, the first and second TALE-nuclease systems regulate the expression of PD-1 and CISH.

[0115] In some embodiments, the first and second TALE-nuclease systems regulate the expression of PD-1 and CBL-B.

[0116] In some embodiments, the first and second TALE-nuclease systems regulate the expression of PD-1 and TIGIT.

[0117] In some embodiments, the first and second TALE-nuclease systems regulate the expression of CTLA-4 and LAG-3.

[0118] In some embodiments, the first and second TALE-nuclease systems regulate the expression of CTLA-4 and CISH.

[0119] In some embodiments, the first and second TALE-nuclease systems regulate the expression of CTLA-4 and CBL-B.

[0120] In some embodiments, the first and second TALE-nuclease systems regulate the expression of LAG-3 and CISH.

[0121] In some embodiments, the first and second TALE-nuclease systems regulate the expression of LAG-3 and CBL-B.

[0122] In some embodiments, the first and second TALE-nuclease systems regulate the expression of CISH and CBL-B.

[0123] In some embodiments, the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein and the second protein are different.

[0124] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CTLA-4.

[0125] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and LAG-3.

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

[0127] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and CBL-B.

[0128] In some embodiments, the first protein and the second protein are selected from the group consisting of PD-1 and TIGIT.

[0129] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and LAG-3.

[0130] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CISH.

[0131] In some embodiments, the first protein and the second protein are selected from the group consisting of CTLA-4 and CBL-B.

[0132] In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CISH.

[0133] In some embodiments, the first protein and the second protein are selected from the group consisting of LAG-3 and CBL-B.

[0134] In some embodiments, the first protein and the second protein are selected from the group consisting of CISH and CBL-B.

[0135] In some embodiments, the first protein is PD-1 and the second protein is CTLA-4.

[0136] In some embodiments, the first protein is CTLA-4 and the second protein is PD-1.

[0137] In some embodiments, the first protein is PD-1 and the second protein is LAG-3.

[0138] In some embodiments, the first protein is LAG-3 and the second protein is PD-1.

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

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

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

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

[0143] In some embodiments, the first protein is PD-1 and the second protein is TIGIT.

[0144] In some embodiments, the first protein is TIGIT and the second protein is PD-1.

[0145] In some embodiments, the first protein is CTLA-4 and the second protein is LAG-3.

[0146] In some embodiments, the first protein is LAG-3 and the second protein is CTLA-4.

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

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

[0149] In some embodiments, the first protein is CTLA-4 and the second protein is CBL-B.

[0150] In some embodiments, the first protein is CBL-B and the second protein is CTLA-4.

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

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

[0153] In some embodiments, the first protein is LAG-3 and the second protein is CBL-B.

[0154] In some embodiments, the first protein is CBL-B and the second protein is LAG-3.

[0155] In some embodiments, the first protein is CISH and the second protein is CBL-B.

[0156] In some embodiments, the first protein is CBL-B and the second protein is CISH.

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

[0158] In some embodiments, the first protein or the second protein is CTLA-4.

[0159] In some embodiments, the first protein or the second protein is LAG-3.

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

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

[0162] In some embodiments, the first protein or the second protein is TIGIT.

[0163] In some embodiments, the first gene editor downregulates expression of a first protein, and the second gene editor downregulates expression of a second protein.

[0164] In some embodiments, the antigen presenting cells (APCs) are PBMCs.

[0165] In some embodiments, the PBMCs are irradiated and allogeneic.

[0166] In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0167] In some embodiments, the IL-2 concentration is about 10,000 IU / mL to about 5,000 IU / mL.

[0168] In some embodiments, the first cell culture medium and / or the second cell culture medium further comprise a 4-1BB agonist and / or an OX40 agonist.

[0169] In some embodiments, the tumor tissue is processed into multiple tumor fragments.

[0170] In some embodiments, the tumor fragments are added to a closed system.

[0171] In some embodiments, no more than 150 fragments, no more than 100 fragments, or no more than 50 fragments are added to the closed system. 167. A gene-edited population of tumor-infiltrating lymphocytes (TILs), comprising an expanded TIL population, wherein expression of at least one protein is modulated by a gene editor transferred into at least a portion of the expanded TIL population.

[0172] In some embodiments, the gene editor is a TALE nuclease system for regulating expression of at least one protein.

[0173] In some embodiments, the at least one protein is PD-1.

[0174] In some embodiments, at least one protein is CTLA-4.

[0175] In some embodiments, at least one protein is LAG-3.

[0176] In some embodiments, at least one protein is CISH.

[0177] In some embodiments, the at least one protein is CBL-B.

[0178] In some embodiments, at least one protein is TIGIT.

[0179] In some embodiments, the expression of at least two proteins is regulated by at least two gene editors introduced into at least a portion of the expanded TIL population, wherein the at least two gene editors comprise a first gene editor comprising a first TALE-nuclease system for regulating expression of a first protein, and a second gene editor comprising a second TALE-nuclease system for regulating expression of a second protein.

[0180] In some embodiments, the first and second proteins are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein and the second protein are different.

[0181] In some embodiments, the first and second proteins are selected from the group consisting of PD-1 and CTLA-4.

[0182] In some embodiments, the first and second proteins are selected from the group consisting of PD-1 and LAG-3.

[0183] In some embodiments, the first and second proteins are selected from the group consisting of PD-1 and CISH.

[0184] In some embodiments, the first and second proteins are selected from the group consisting of PD-1 and CBL-B.

[0185] In some embodiments, the first and second proteins are selected from the group consisting of PD-1 and TIGIT.

[0186] In some embodiments, the first and second proteins are selected from the group consisting of CTLA-4 and LAG-3.

[0187] In some embodiments, the first and second proteins are selected from the group consisting of CTLA-4 and CISH.

[0188] In some embodiments, the first and second proteins are selected from the group consisting of CTLA-4 and CBL-B.

[0189] In some embodiments, the first and second TALE proteins are selected from the group consisting of LAG-3 and CISH.

[0190] In some embodiments, the first and second proteins are selected from the group consisting of LAG-3 and CBL-B.

[0191] In some embodiments, the first and second proteins are selected from the group consisting of CISH and CBL-B.

[0192] In some embodiments, the gene-edited TIL populations disclosed herein are produced by the methods disclosed herein.

[0193] In some embodiments, provided herein is a pharmaceutical composition comprising a gene-edited TIL population disclosed herein and a pharmaceutically acceptable carrier.

[0194] In some embodiments, provided herein is a method for treating a subject having cancer, the method comprising administering a therapeutically effective dose of a gene-edited TIL population disclosed herein.

[0195] In some embodiments, the cancer is selected from the group consisting of melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

[0196] In some embodiments, described herein are methods for treating a subject having cancer, the methods 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 the patient into a plurality of tumor fragments; (b) performing a first expansion by adding the tumor fragments to a closed system and culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 8 days to obtain the second TIL population; (c) activating the second TIL population for 1 to 6 days using anti-CD3 agonist beads or antibodies, or anti-CD3 and anti-CD28 agonist beads or antibodies, to produce a third TIL population; (e) performing a sterile electroporation step on the third population of TILs, wherein the sterile electroporation step mediates the transfer of at least one gene editor; (f) allowing the third population of TILs to rest for about 1 day; (g) performing a second expansion of the third TIL population by culturing it in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a fourth TIL population, wherein the second expansion is performed for about 5 to 15 days to obtain the third TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, wherein the fourth TIL population is a therapeutic TIL population; and (h) harvesting the therapeutic TIL population obtained from step (e) to provide a harvested TIL population, wherein one or more of steps (a) through (h) are performed in a closed, sterile system; (i) transferring the harvested TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) cryopreserving the harvested TIL population using a dimethyl sulfoxide-based cryopreservation medium; and (k) administering a therapeutically effective dose of the harvested population of TILs from the infusion bag to the patient; The electroporation step includes delivery of a transcription activator-like effector nuclease (TALEN) system to inhibit expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT and / or CBL-B.

[0197] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of PD-1.

[0198] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CTLA-4.

[0199] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of LAG-3.

[0200] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CISH.

[0201] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CBL-B.

[0202] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of TIGIT.

[0203] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of PD-1 and CTLA-4.

[0204] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of PD-1 and LAG-3.

[0205] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of PD-1 and CISH.

[0206] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of PD-1 and CBL-B.

[0207] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of PD-1 and TIGIT.

[0208] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CTLA-4 and LAG-3.

[0209] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CTLA-4 and CISH.

[0210] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CTLA-4 and CBL-B.

[0211] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CTLA-4 and TIGIT.

[0212] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of LAG-3 and CISH.

[0213] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of LAG-3 and CBL-B.

[0214] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of LAG-3 and TIGIT.

[0215] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CISH and CBL-B.

[0216] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CISH and TIGIT.

[0217] In some embodiments, the electroporation step includes delivery of a TALEN system to inhibit expression of CBL-B and TIGIT.

[0218] In some embodiments, the therapeutically effective dose of TILs is about 1×10 9 ~Approx. 1×10 11 It's TIL.

[0219] In some embodiments, a non-myeloablative lymphodepletion regimen has been administered to the patient prior to administering the therapeutically effective dose of the harvested TIL population in step (k).

[0220] In some embodiments, the method further comprises treating the patient with a high-dose IL-2 regimen beginning the day after administering a therapeutically effective dose of the harvested population of TILs to the patient in step (k).

[0221] In some embodiments, the cancer is selected from the group consisting of melanoma, metastatic melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), metastatic NSCLC, lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

[0222] In some embodiments, the cancer is melanoma.

[0223] In some embodiments, the cancer is metastatic melanoma.

[0224] In some embodiments, the cancer is NSCLC.

[0225] In some embodiments, the cancer is metastatic NSCLC.

[0226] In some embodiments, the gene editing silences or reduces expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. [Brief explanation of the drawings]

[0227] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 3] FIG. 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] FIG. 1 shows a diagram of an embodiment of Gen2 (Process 2A), a 22-day process for TIL fabrication. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1. Exemplary Gen3 TIL fabrication process. [Figure 8A] A comparison of embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication is shown. [Figure 8B]An exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14- to 16-day processes) for each of the three process variations. [Figure 8D] An exemplary modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 8E] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8F] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8G] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8H] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8I] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8J] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8K] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8L] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8M] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8N] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8O] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 8P] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10]1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17] Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18] Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from antibodies that bind, for example, to 4-1BBL or 4-1BB, that fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together via disulfide bonds (small oblong ellipses), stabilizing the structure and providing an agonist that can bring together the six receptor and intracellular signaling domains of the signaling protein to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19]Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 testing process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] Schematic of an exemplary embodiment of the preparation timeline for the Gen3 process (16-17 day process). [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 33] Approval criteria table. [Figure 34] Experimental flow diagram of the full-scale PD-1 KO TIL TALEN process. [Figure 35] Experimental flow diagram of the full-scale PD-1 KO TIL TALEN process. [Figure 36] Schematic of an exemplary embodiment of the KO TIL TALEN process. [Figure 37] Schematic of an exemplary embodiment of the process described in Example 12. [Figure 38] In vivo efficacy of PDCD-1 KO TILs. A) Efficacy of PDCD-1 KO assessed by flow cytometry. B) hIL-2 NOG mice (n=14 per treatment group) engrafted with melanoma tumor cells were adoptively transferred with PDCD-1 KO or mock TILs. Anti-PD-1 antibody treatment in combination with mock TILs was included as a control for PD-1 / PD-L1 blockade. Statistical significance is indicated by *p<0.05, **p<0.01, and ****p<0.0001. [Figure 39] Analysis of TIL products. A) Viable cell dose, B) Purity, C) Identity, D) Potency, and E) PDCD-1 KO efficiency of TIL products. [Figure 40] Analysis of TIL products. A) TIL differentiation, and B) TIL memory. [Figure 41] Expression of activation- and inhibition-associated markers on PDCD-1 KO TILs. [Figure 42] IL-2-independent proliferation assay of PDCD-1 KO TIL products. [Figure 43] Summary of karyotyping results from PDCD-1 KO TIL products. [Figure 44] Cell viability (A) and fold recovery (B) of cells before electroporation. [Figure 45] Magnification recovery of cells after electroporation (A) and cell viability (B). [Figure 46] Knockout efficiency in CD3+ (A), CD8+ (B), and CD4+ (C) cells. [Figure 47] Magnification recovery of cells after electroporation (A) and cell viability (B). [Figure 48]Fold recovery of cells (A) and cell viability (B) after electroporation using 6000 IU / mL of IL-2. [Figure 49] Fold recovery of cells (A) and cell viability (B) after electroporation using various conditions. [Figure 50] Knockout efficiency in CD3+ (A), CD8+ (B), and CD4+ (C) cells. [Figure 51] Cell viability before electroporation. [Figure 52] Magnification recovery of cells before electroporation. [Figure 53] Magnification recovery of cells after electroporation (A) and cell viability (B). [Figure 54A] Knockout efficiency in CD3+ cells. [Figure 54B] Knockout efficiency in CD8+ cells. [Figure 54C] Knockout efficiency in CD4+ cells. [Figure 55] Cell number (A) and viability (B) after various washing steps. [Figure 56] Cell counts after various spin conditions using a PBS wash (A) or a Cyto wash (B). [Figure 57] Cell viability after various spin conditions using a PBS wash (A) or a Cyto wash (B). [Figure 58] Total spin-relative cell number (A) and total spin-relative cell viability (B) of cells after various spin conditions. [Figure 59] Total spin comparative cell loss rate after various spin conditions. [Figure 60] Loss and viability during electroporation, specifically, cell loss during the washing step (A), cell loss after electroporation (B), and cell viability after electroporation. [Figure 61] Knockout efficiency in CD3+ (A), CD8+ (B), and CD4+ (C) cells. [Figure 62]Cell viability (A) and fold expansion (B) of REP harvests. [Figure 63] Cell loss rate (A) and cell viability (B) after electroporation. [Figure 64] Knockout efficiency in CD3+ (A), CD4+ (B), and CD8+ (C) cells. [Figure 65] Fold expansion (A) and cell viability (B) of REP harvest. [Figure 66] Cell proliferation (A), first electroporation knockout efficiency (B), and second electroporation knockout efficiency (C). [Figure 67] Growth rate during 3 days of standing. [Figure 68] PD-1 knockout efficiency. [Figure 69] PDCD1 gene modification by NGS. [Figure 70] Distribution of TCR Vβ subtypes in bulk PD-1 KO TIL products and distribution of NE TILs in the CD3+PD-1 subset. [Figure 71] PD-1 KO TIL effector function as measured by MLR (A) and multifunctionality (B). [Figure 72] In vivo antitumor activity of M1152 PD-1 KO TIL products. [Figure 73] Persistence of TALEN proteins in autologous TILs as a function of time measured by Western blot. [Figure 74A] Illustrative TIL fabrication process. [Figure 74B] Illustrative TIL fabrication process. [Figure 74C] Illustrative TIL fabrication process. [Fig. 74D] Illustrative TIL fabrication process. [Figure 74E] Illustrative TIL fabrication process. [Figure 74F] Illustrative TIL fabrication process. [Figure 75A] Schema of the Phase 1 / 2 study described in Example 22. [Figure 75B] Schema of the Phase 1 / 2 study described in Example 22. [Figure 76] Summary of data described in Example 23. [Figure 77] Results of the demo day experiment for Example 23. [Figure 78A] Results from Example 23, Neon Exp 1. [Figure 78B] Results from Example 23, Neon Exp 1. [Figure 78C] Results from Example 23, Neon Exp 1. [Figure 79] Results from Example 23, Xenon Exp 1. [Figure 80] Results for Example 23, Xenon Exp 3. [Figure 81] Results for Example 23, Xenon Exp 4.

[0228] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0229] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0230] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0231] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0232] SEQ ID NO: 5 is an IL-2 form.

[0233] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0234] SEQ ID NO: 7 is an IL-2 form.

[0235] SEQ ID NO: 8 is a mucin domain polypeptide.

[0236] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0237] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0238] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0239] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0240] SEQ ID NO: 13 is the IL-2 sequence.

[0241] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0242] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0243] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0244] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0245] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0246] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0247] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0248] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0249] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0250] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0251] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0252] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0253] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0254] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0255] SEQ ID NO: 28 is the V H It's a chain.

[0256] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0257] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0258] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0259] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0260] SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

[0261] SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

[0262] SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

[0263] SEQ ID NO: 36 is V L It's a chain.

[0264] SEQ ID NO: 37 is the light chain.

[0265] SEQ ID NO: 38 is the light chain.

[0266] SEQ ID NO: 39 is the light chain.

[0267] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0268] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0269] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0270] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0271] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0272] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0273] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0274] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0275] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0276] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0277] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0278] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0279] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0280] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0281] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0282] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0283] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0284] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0285] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0286] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0287] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0288] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0289] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0290] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0291] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0292] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0293] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0294] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0295] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0296] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0297] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0298] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0299] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0300] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0301] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0302] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0303] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0304] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0305] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0306] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0307] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0308] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0309] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0310] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0311] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0312] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0313] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0314] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0315] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0316] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H )

[0317] SEQ ID NO: 90 is the light chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). L )

[0318] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0319] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0320] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0321] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0322] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0323] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0324] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0325] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0326] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0327] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0328] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0329] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0330] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0331] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0332] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0333] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0334] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0335] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0336] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0337] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0338] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0339] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0340] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0341] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0342] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0343] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0344] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0345] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0346] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0347] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0348] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0349] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0350] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0351] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0352] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0353] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0354] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0355] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0356] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0357] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0358] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0359] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0360] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0361] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0362] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0363] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0364] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0365] SEQ ID NO: 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0366] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0367] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0368] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0369] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0370] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0371] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0372] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0373] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0374] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0375] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0376] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0377] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0378] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0379] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0380] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0381] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0382] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0383] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0384] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0385] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0386] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0387] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0388] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0389] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0390] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0391] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0392] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0393] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0394] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0395] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0396] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0397] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0398] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0399] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0400] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0401] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0402] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0403] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0404] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0405] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0406] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0407] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0408] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0409] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0410] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0411] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0412] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0413] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0414] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0415] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0416] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0417] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0418] SEQ ID NO: 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0419] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0420] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0421] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0422] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0423] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0424] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0425] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0426] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0427] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0428] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0429] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0430] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0431] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0432] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0433] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0434] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0435] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0436] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0437] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0438] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0439] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0440] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0441] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0442] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0443] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0444] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0445] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0446] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0447] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0448] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0449] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0450] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0451] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0452] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0453] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0454] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0455] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0456] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0457] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0458] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0459] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0460] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0461] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0462] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0463] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0464] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0465] SEQ ID NO: 238 is an exemplary Clo051 nuclease domain amino acid sequence.

[0466] SEQ ID NO: 239 is an exemplary piggyBac (PB) transposase enzyme amino acid sequence.

[0467] SEQ ID NO:240 is an exemplary Sleeping Beauty transposase enzyme amino acid sequence.

[0468] SEQ ID NO: 241 is an exemplary hyperactive Sleeping Beauty (SB100X) transposase amino acid sequence.

[0469] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referenced herein are incorporated by reference in their entirety.

[0470] As used herein, the terms "co-administration," "co-administering," "administered in combination," "administering in combination," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs in preferred embodiments of the present invention) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Simultaneous administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0471] The term "in vivo" refers to events that take place inside a subject's body.

[0472] The term "in vitro" refers to events that occur outside a subject's body. In vitro assays include cell-based assays, in which living or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0473] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic manner.

[0474] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, 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 described herein.

[0475] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations 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.

[0476] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically yields a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0477] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded (REP TILs), that are processed and stored at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0478] By "thawed cryopreserved TILs" herein is meant a population of TILs that have been previously cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs can be administered to a patient.

[0479] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0480] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium that contains DMSO. In some embodiments, CS10 medium contains 10% DMSO.

[0481] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高) is a subset of T cells that constitutively express the CD4 receptor. 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. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0482] The term "effector memory T cells" refers to T cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低 ), refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon gamma, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0483] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, sealed G containers. Once tumor segments are added to the closed system, the system is not opened to the external environment until the TILs are ready to be administered to a patient.

[0484] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0485] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0486] The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0487] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0488] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0489] [Table 1]

[0490] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug bempegaldesleukin (NKTR-214, an IL-2 prodrug in which an average of six lysine residues are replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6The present invention also encompasses pegylated forms of IL-2 described herein, including pegylated human recombinant IL-2 such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, CA, USA) or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. US2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Benpegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791 A1 and International Patent Application Publication No. WO2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0491] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 are ...and Y107 are further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid is selected from N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 complex has a reduced affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than a 99% reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, or greater reduction in binding affinity for IL-2Rα compared to a wild-type IL-2 polypeptide.1000-fold or more. In some embodiments, the conjugated moiety impairs or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water soluble polymers independently comprises a glycan. In some embodiments, each of the water soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moieties comprise a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moieties comprise a polypeptide. In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP),The conjugated moiety may comprise a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugated moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugated moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-(3'-(2'-pyridyldithio) (e)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker isIn some embodiments, the heterobifunctional linker includes N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexamethyl. Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimide Succinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(( (iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4, -azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfo Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)- 1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl diazo-3 ,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimide group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any of the IL-2 forms described herein. In some embodiments, IL-2 forms suitable for use in the present invention are pegylated as disclosed in U.S. Patent Application Publication Nos. US2020 / 0181220 A1 and US2020 / 0330601 A1. In some embodiments, IL-2 forms suitable for use in the present invention are IL-2 conjugates comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5.

[0492] In some embodiments, a form of IL-2 suitable for use in the present invention is nembareukin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembareukin alfa is a nucleotide analogue of IL-2 that is linked via a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS138 Human interleukin-2 receptor α-chain fragment (139-303) fused via a nucleotide sequence (Cys), produced in Chinese hamster ovary (CHO) cells, and glycosylated. 125 >Ser 51 ; human interleukin-2 (IL-2) (4-74)-peptide (62-132) fused via a G2 peptide linker (60-61) and human interleukin-2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) fused via a GSG3S peptide linker (133-138), produced in Chinese hamster ovary (CHO) cells, and alpha-glycosylated human interleukin-2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nemvaleukin alfa is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering of SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212, using the numbering of SEQ ID NO: 6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. US2021 / 0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity for IL-Rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to the fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

[0493] [Table 2-1] [Table 2-2]

[0494] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDR of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V Land an IL-2 molecule or fragment thereof grafted onto the CDRs of, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. 2020 / 0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine grafted protein comprises a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and a VL comprising complementarity determining regions HCDR1, HCDR2, and LCDR3. H or V L or a fragment thereof grafted onto the CDR of the antibody cytokine grafted protein, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells, and the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 29, an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 38.

[0495] In some embodiments, the IL-2 molecule or fragment thereof is V H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. L In some embodiments, the IL-2 molecule or a fragment thereof is grafted onto LCDR1 of V LIn some embodiments, the IL-2 molecule or fragment thereof is grafted onto LCDR2 of V L The LCDR3 of the IL-2 molecule is grafted onto the LCDR3 of the IL-2 molecule, and the IL-2 molecule is a mutein.

[0496] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0497] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0498] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. US2020 / 0270334 A1, the disclosure of which is incorporated herein by reference.

[0499] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H In some embodiments, the antibody cytokine transplant protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 36. L In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 28. H V comprising the region and the amino acid sequence of SEQ ID NO: 36 LIn some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or a variant, derivative, or fragment thereof, or conservative amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant proteins described herein comprises immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or an equivalent molecule. In some embodiments, the antibody cytokine transplant proteins described herein have a sequence set forth in Table 3.

[0500] [Table 3-1] [Table 3-2] [Table 3-3]

[0501] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0502] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin-7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, which is a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of a recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0503] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 11).

[0504] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 primarily stimulates natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 21).

[0505] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, the tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg of body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10The TIL (optionally including genetically engineered TIL) compositions may be administered at doses of 1000-15000 cells (including all integer values ​​within those ranges). TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these doses. TIL (optionally including genetically engineered TIL) can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). Optimal dosages and treatment regimes for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0506] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0507] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MILs, TILs, and PBLs are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0508] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0509] In some embodiments, the invention includes methods of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27-23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0510] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0511] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including, but not limited to, disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and condition being treated (e.g., the subject's weight, age, and sex), the severity of the condition, or the method of administration. The term also applies to a dose that induces a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0512] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also 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 pathology, e.g., in the case of a vaccine.

[0513] "Non-myelinating chemotherapy," "non-myelinating lymphodepleting," "NMALD," "NMA LD," "NMA-LD," and any variants of the foregoing, are used interchangeably to refer to chemotherapy regimens designed to deplete a patient's lymphoid immune cells while avoiding depletion of a patient's myeloid immune cells. Typically, a patient undergoes a course of non-myeloablative chemotherapy before administering tumor-infiltrating lymphocytes to the patient, as described herein.

[0514] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0515] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0516] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0517] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0518] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0519] The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, the base moiety, and / or the linkages between deoxyribonucleotides in an oligonucleotide.

[0520] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0521] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0522] The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0523] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional, unrecited claim elements or steps, if any, are excluded from the scope of the claim(s). The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0524] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0525] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound to a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen preferably reacts with a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0526] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0527] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL and (v) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, which can be synthesized using recombinant methods. L and V H The regions may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L If the domain is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV L Methods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0528] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0529] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0530] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0531] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0532] The phrases "an antibody that recognizes an antigen" and "an antibody that is specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0533] The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of the antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0534] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0535] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0536] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described, for example, in European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0537] The term "glycosylation" refers to modified derivatives of antibodies. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes can be used to cleave fucose residues from antibodies. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies, as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0538] "PEGylation" refers to a modified antibody or fragment thereof that has been reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol, or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. EP 0 154 316 and EP 0 401 384, and U.S. Pat. No. 5,824,778 (the disclosures of each of which are incorporated herein by reference).

[0539] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, including monoclonal antibodies or proteins, and that has no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (proleukin), a protein approved by a drug regulatory agency for aldesleukin is a "biosimilar" of aldesleukin or a "biosimilar of" aldesleukin. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, biosimilars may be authorized or approved for authorisation or licensing under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared in specific studies to biopharmaceuticals authorized outside the European Economic Area (non-EEA authorized "comparators"). Such studies include, for example, specific clinical studies and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may have an amino acid sequence that has 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference pharmaceutical. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from the post-translational modifications of the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical. A biosimilar may have the same or a different glycosylation pattern as the reference pharmaceutical, provided that the differences do not result in a change in the safety and / or efficacy of the pharmaceutical.In particular, but not exclusively, biosimilars may have different glycosylation patterns if the differences address or are intended to address safety concerns associated with the reference drug. Additionally, biosimilars may deviate from the reference drug, for example, in its strength, dosage form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain differences, for example, in their pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles compared to the reference drug, but are still considered sufficiently similar to the reference drug to be approved or deemed suitable for approval. In certain circumstances, biosimilars exhibit different binding characteristics compared to the reference drug, and these different binding characteristics are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0540] II. Gene Editing Process A. Overview: TIL expansion + gene editing

[0003] Embodiments of the present invention relate to methods for expanding a TIL population, the methods comprising one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, "gene editing," "gene editing," and "genome editing" refer to types of genetic modification in which DNA is permanently modified within a cell's genome, for example, DNA is inserted, deleted, modified, or replaced within the cell's genome. In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockdown) the expression of a DNA sequence. According to embodiments of the present invention, gene editing techniques are used to enhance the efficacy of a therapeutic population of TILs.

[0541] The method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the embodiments of the methods described herein, where the method further comprises gene editing at least a portion of the TILs. According to further embodiments, the method for expanding TILs into a therapeutic TIL population is carried out according to any of the embodiments of the methods described in WO2018 / 081473A1, WO2018 / 129332A1, or WO2018 / 182817A1 (which are incorporated by reference in their entireties), where the method further comprises gene editing at least a portion of the TILs. Thus, certain embodiments of the present invention provide a therapeutic TIL population expanded according to any of the embodiments described herein, where at least a portion of the therapeutic population is gene edited, e.g., at least a portion of the therapeutic TIL population transferred to an infusion bag is permanently gene edited.

[0542] B. Gene editing during TIL expansion In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population for 1 to 7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) culturing the fourth TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0543] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 9 days to produce a second TIL population; (d) activating the second TIL population for 1 to 7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (e) gene editing at least a portion of the third TIL population to produce a fourth TIL population; (f) culturing the fourth TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0544] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3-9 days to produce a second TIL population; (c) activating the second TIL population for 1 to 7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) culturing the fourth TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 1 to 7 days to produce a culture of a fifth TIL population; (f) dividing the culture of the fifth TIL population into a plurality of subcultures, culturing each of the plurality of subcultures in a third cell culture medium containing IL-2 for about 3 to 7 days, and combining the plurality of subcultures to provide an expanded number of TILs.

[0545] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 to 9 days to produce a second TIL population; (d) activating the second TIL population for 1 to 7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (e) gene editing at least a portion of the third TIL population to produce a fourth TIL population; (f) culturing the fourth TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 1 to 7 days to produce a culture of a fifth TIL population; (g) dividing the culture of the fifth TIL population into multiple subcultures, culturing each of the multiple subcultures for about 3 to 7 days in a third cell culture medium containing IL-2, and combining the multiple subcultures to provide an expanded number of TILs.

[0546] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) culturing a first population of TILs obtained by digesting tumor tissue excised from a subject or patient in an enzymatic medium to produce a tumor digest in a first cell culture medium containing IL-2 and OKT-3 for about 3 to 9 days to produce a second population of TILs; (b) gene editing at least a portion of the second TIL population to produce a third TIL population; (c) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0547] In some embodiments, the method comprises culturing or initially expanding the first TIL population in a first cell culture medium comprising IL-2 for about 3 days, followed by culturing the first TIL population in a cell culture medium comprising IL-2 and OKT-3 for 2-6 days.

[0548] In some embodiments, the method includes the step of culturing or rapidly expanding a third TIL population by culturing the third TIL population in a second cell culture medium for a first period of about 1 to 7 days, wherein at the end of the first period, the culture is divided into multiple subcultures, and each of the multiple subcultures is cultured in a third culture medium containing IL-2 for a second period of about 3 to 7 days, and wherein at the end of the second period, the multiple subcultures are combined to provide an expanded number of TILs.

[0549] In some embodiments, culturing the first TIL population is performed for about 3-9 days. In some embodiments, culturing the first TIL population is performed for about 3-9 days, about 3-8 days, about 4-8 days, about 5-8 days, about 6-8 days, about 7-8 days, about 3-7 days, about 4-7 days, about 5-7 days, about 6-7 days, about 3-6 days, about 4-6 days, about 5-6 days, about 3-5 days, about 4-5 days, or about 3-4 days. In some embodiments, culturing the first TIL population is performed for about 3 days. In some embodiments, culturing the first TIL population is performed for about 4 days. In some embodiments, culturing the first TIL population is performed for about 5 days. In some embodiments, culturing the first TIL population is performed for about 6 days. In some embodiments, culturing the first TIL population is performed for about 7 days. In some embodiments, culturing the first TIL population is carried out for about 8 days. In some embodiments, culturing the first TIL population is carried out for about 9 days.

[0550] In some embodiments, the step of activating the second TIL population is carried out for about 1 to 7 days. In some embodiments, the step of activating the second TIL population is carried out for about 1 to 7 days, about 1 to 6 days, about 2 to 6 days, about 3 to 6 days, about 4 to 6 days, about 5 to 6 days, about 1 to 5 days, about 2 to 5 days, about 3 to 5 days, about 4 to 5 days, about 1 to 4 days, about 2 to 4 days, about 3 to 4 days, about 1 to 3 days, about 2 to 3 days, or about 1 to 2 days. In some embodiments, the step of activating the second TIL population is carried out for about 1 day. In some embodiments, the step of activating the second TIL population is carried out for about 2 days. In some embodiments, the step of activating the second TIL population is carried out for about 3 days. In some embodiments, the step of activating the second TIL population is carried out for about 4 days. In some embodiments, the step of activating the second TIL population is carried out for about 5 days. In some embodiments, the step of activating the second TIL population is carried out for about 6 days. In some embodiments, the step of activating the second TIL population is carried out for about 7 days.

[0551] In some embodiments, culturing the fourth TIL population is carried out for about 5 to 15 days. In some embodiments, culturing the fourth TIL population is carried out for about 5 to 15 days, about 6 to 15 days, about 7 to 15 days, about 8 to 15 days, about 9 to 15 days, about 10 to 15 days, about 11 to 15 days, about 12 to 15 days, about 13 to 15 days, about 14 to 15 days, about 5 to 14 days, about 6 to 14 days, about 7 to 14 days, about 8 to 14 days, about 9 to 14 days, about 10 to 14 days, about 11 to 14 days, about 12 to 14 days, about 13 to 14 days, about 5 to 13 days, about 6 to 13 days, about 7 to 13 days, about 8 to 13 days, about 9 to 13 days, about 10 to 15 days. In some embodiments, culturing the fourth TIL population is performed for about 13 days, about 11-13 days, about 12-13 days, about 5-12 days, about 6-12 days, about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, about 11-12 days, about 5-11 days, 6-11 days, 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 5-10 days, 6-10 days, 7-10 days, about 8-10 days, about 9-10 days, about 5-9 days, 6-9 days, 7-9 days, about 8-9 days, about 5-8 days, about 6-8 days, 7-8 days, about 5-7 days, about 6-7 days, or about 5-6 days. In some embodiments, culturing the fourth TIL population is performed for about 5 days. In some embodiments, culturing the fourth TIL population is performed for about 6 days. In some embodiments, culturing the fourth TIL population is performed for about 7 days. In some embodiments, culturing the fourth TIL population is performed for about 8 days. In some embodiments, culturing the fourth TIL population is performed for about 9 days. In some embodiments, culturing the fourth TIL population is performed for about 10 days. In some embodiments, culturing the fourth TIL population is performed for about 11 days. In some embodiments, culturing the fourth TIL population is performed for about 12 days. In some embodiments, culturing the fourth TIL population is performed for about 13 days. In some embodiments, culturing the fourth TIL population is performed for about 14 days. In some embodiments, culturing the fourth TIL population is performed for about 15 days.

[0552] In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 8 days. In some embodiments, the steps of this method are completed within a time period of about 9 days. In some embodiments, the steps of this method are completed within a time period of about 10 days. In some embodiments, the steps of this method are completed within a time period of about 11 days. In some embodiments, the steps of this method are completed within a time period of about 12 days. In some embodiments, the steps of this method are completed within a time period of about 13 days. In some embodiments, the steps of this method are completed within a time period of about 14 days. In some embodiments, the steps of this method are completed within a time period of about 15 days. In some embodiments, the steps of this method are completed within a time period of about 16 days. In some embodiments, the steps of this method are completed within a time period of about 17 days. In some embodiments, the steps of this method are completed within a time period of about 18 days. In some embodiments, the steps of this method are completed within a time period of about 19 days. In some embodiments, the steps of this method are completed within a time period of about 20 days. In some embodiments, the steps of this method are completed within a time period of about 21 days. In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 23 days. In some embodiments, the steps of this method are completed within a time period of about 24 days. In some embodiments, the steps of this method are completed within a time period of about 25 days. In some embodiments, the steps of this method are completed within a time period of about 26 days. In some embodiments, the steps of this method are completed within a time period of about 27 days. In some embodiments, the steps of this method are completed within a time period of about 28 days. In some embodiments, the steps of this method are completed within a time period of about 29 days. In some embodiments, the steps of this method are completed within a time period of about 30 days. In some embodiments, the steps of this method are completed within a time period of about 31 days.

[0553] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0554] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0555] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0556] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0557] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0558] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0559] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0560] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0561] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0562] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0563] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0564] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0565] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0566] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0567] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0568] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0569] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0570] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0571] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0572] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0573] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0574] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0575] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0576] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0577] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0578] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0579] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0580] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0581] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0582] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0583] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0584] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0585] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0586] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0587] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0588] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0589] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0590] In some embodiments, the gene editing process can be performed at any time during the TIL expansion method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, between any of steps (a)-(e), (a)-(f), or (a)-(g) outlined in the method above, or before or after any of steps (a)-(e), (a)-(f), or (a)-(g) outlined in the method above. In some embodiments, the gene editing process can be performed more than once at any time during the TIL expansion method. According to certain embodiments, the TILs are collected during the culture step (e.g., the culture step is "paused" for at least some of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the culture step (e.g., returned to culture medium) to continue the culture step, thereby permanently gene editing at least some of the therapeutic TIL population that will ultimately be transferred to the infusion bag.

[0591] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(e), (a)-(f), or (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 more than two culture steps, and gene editing may be performed on the TILs during the third or fourth culture step, etc.

[0592] According to some embodiments, gene editing is performed while the TILs are still in the culture medium and while the culturing step is being performed, i.e., they are not necessarily "removed" from the culture step to perform the gene editing. According to some embodiments, gene editing is performed on TILs that have been harvested from the culture medium, and after the gene editing process, the TILs are then returned to the culture medium.

[0593] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3 to 9 days to produce a second TIL population; (c) gene editing at least a portion of the second TIL population to produce a third TIL population; (d) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0594] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3 to 9 days to produce a second TIL population; (d) gene editing at least a portion of the second TIL population to produce a third TIL population; (e) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0595] In some embodiments, culturing the first TIL population is performed for about 3-9 days. In some embodiments, culturing the first TIL population is performed for about 3-9 days, about 3-8 days, about 4-8 days, about 5-8 days, about 6-8 days, about 7-8 days, about 3-7 days, about 4-7 days, about 5-7 days, about 6-7 days, about 3-6 days, about 4-6 days, about 5-6 days, about 3-5 days, about 4-5 days, or about 3-4 days. In some embodiments, culturing the first TIL population is performed for about 3 days. In some embodiments, culturing the first TIL population is performed for about 4 days. In some embodiments, culturing the first TIL population is performed for 5 days. In some embodiments, culturing the first TIL population is performed for about 6 days. In some embodiments, culturing the first TIL population is performed for about 7 days. In some embodiments, culturing the first TIL population is carried out for about 8 days. In some embodiments, culturing the first TIL population is carried out for about 9 days.

[0596] In some embodiments, culturing the third TIL population is carried out for about 5 to 15 days. In some embodiments, culturing the third TIL population is carried out for about 5 to 15 days, about 6 to 15 days, about 7 to 15 days, about 8 to 15 days, about 9 to 15 days, about 10 to 15 days, about 11 to 15 days, about 12 to 15 days, about 13 to 15 days, about 14 to 15 days, about 5 to 14 days, about 6 to 14 days, about 7 to 14 days, about 8 to 14 days, about 9 to 14 days, about 10 to 14 days, about 11 to 14 days, about 12 to 14 days, about 13 to 14 days, about 5 to 13 days, about 6 to 13 days, about 7 to 13 days, about 8 to 13 days, about 9 to 13 days, about 10 to 15 days. In some embodiments, culturing the third TIL population is performed for about 13 days, about 11-13 days, about 12-13 days, about 5-12 days, about 6-12 days, about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, about 11-12 days, about 5-11 days, 6-11 days, 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 5-10 days, 6-10 days, 7-10 days, about 8-10 days, about 9-10 days, about 5-9 days, 6-9 days, 7-9 days, about 8-9 days, about 5-8 days, about 6-8 days, 7-8 days, about 5-7 days, about 6-7 days, or about 5-6 days. In some embodiments, culturing the third TIL population is performed for about 5 days. In some embodiments, culturing the third TIL population is performed for about 6 days. In some embodiments, culturing the third TIL population is performed for about 7 days. In some embodiments, culturing the third TIL population is performed for about 8 days. In some embodiments, culturing the third TIL population is performed for about 9 days. In some embodiments, culturing the third TIL population is performed for about 10 days. In some embodiments, culturing the third TIL population is performed for about 11 days. In some embodiments, culturing the third TIL population is performed for about 12 days. In some embodiments, culturing the third TIL population is performed for about 13 days. In some embodiments, culturing the third TIL population is performed for about 14 days. In some embodiments, culturing the third TIL population is performed for about 15 days.

[0597] In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 8 days. In some embodiments, the steps of this method are completed within a time period of about 9 days. In some embodiments, the steps of this method are completed within a time period of about 10 days. In some embodiments, the steps of this method are completed within a time period of about 11 days. In some embodiments, the steps of this method are completed within a time period of about 12 days. In some embodiments, the steps of this method are completed within a time period of about 13 days. In some embodiments, the steps of this method are completed within a time period of about 14 days. In some embodiments, the steps of this method are completed within a time period of about 15 days. In some embodiments, the steps of this method are completed within a time period of about 16 days. In some embodiments, the steps of this method are completed within a time period of about 17 days. In some embodiments, the steps of this method are completed within a time period of about 18 days. In some embodiments, the steps of this method are completed within a time period of about 19 days. In some embodiments, the steps of this method are completed within a time period of about 20 days. In some embodiments, the steps of this method are completed within a period of about 21 days. In some embodiments, the steps of this method are completed within a period of about 22 days. In some embodiments, the steps of this method are completed within a period of about 23 days. In some embodiments, the steps of this method are completed within a period of about 24 days.

[0598] In some embodiments, the step of culturing the third TIL population is carried out by culturing the third TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0599] In some embodiments, the step of culturing the third TIL population is carried out by culturing the third TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0600] In some embodiments, the gene editing process can be performed at any time during the TIL expansion method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, between any of steps (a)-(d) or (a)-(e) outlined in the method above, or before or after any of steps (a)-(d) or (a)-(e) outlined in the method above. In some embodiments, the gene editing process can be performed more than once at any time during the TIL expansion method. According to certain embodiments, the TILs are collected during the culture step (e.g., the culture step is "paused" for at least some of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the culture step (e.g., returned to culture medium) to continue the culture step, thereby permanently gene editing at least some of the therapeutic TIL population that will ultimately be transferred to the infusion bag.

[0601] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(d) or (a)-(e), or may have a different number of steps. Regardless of the specific embodiment, the gene editing process may be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two culture steps, and gene editing may be performed on the TIL during the third or fourth culture step, etc.

[0602] According to some embodiments, gene editing is performed while the TILs are still in the culture medium and while the culturing step is being performed, i.e., they are not necessarily "removed" from the culture step to perform the gene editing. According to some embodiments, gene editing is performed on TILs that have been harvested from the culture medium, and after the gene editing process, the TILs are then returned to the culture medium.

[0603] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3 to 9 days to produce a second TIL population; (c) gene editing at least a portion of the second TIL population to produce a third TIL population; (d) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 1 to 7 days to produce a culture of a fourth TIL population; (e) dividing the culture of the fourth TIL population into a plurality of subcultures, culturing each of the plurality of subcultures in a third cell culture medium containing IL-2 for about 3 to 7 days, and combining the plurality of subcultures to provide a fifth TIL population comprising an expanded number of TILs.

[0604] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium comprising IL-2 and OKT-3 for about 3 to 9 days to produce a second TIL population; (d) gene editing at least a portion of the second TIL population to produce a third TIL population; (e) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 1 to 7 days to produce a culture of a fourth TIL population; (f) dividing the culture of the fourth TIL population into a plurality of subcultures, culturing each of the plurality of subcultures in a third cell culture medium containing IL-2 for about 3 to 7 days, and combining the plurality of subcultures to provide a fifth TIL population comprising an expanded number of TILs.

[0605] In some embodiments, culturing the first TIL population is performed for about 3-9 days. In some embodiments, culturing the first TIL population is performed for about 3-9 days, about 3-8 days, about 4-8 days, about 5-8 days, about 6-8 days, about 7-8 days, about 3-7 days, about 4-7 days, about 5-7 days, about 6-7 days, about 3-6 days, about 4-6 days, about 5-6 days, about 3-5 days, about 4-5 days, or about 3-4 days. In some embodiments, culturing the first TIL population is performed for about 3 days. In some embodiments, culturing the first TIL population is performed for about 4 days. In some embodiments, culturing the first TIL population is performed for about 5 days. In some embodiments, culturing the first TIL population is performed for about 6 days. In some embodiments, culturing the first TIL population is performed for about 7 days. In some embodiments, culturing the first TIL population is carried out for about 8 days. In some embodiments, culturing the first TIL population is carried out for about 9 days.

[0606] In some embodiments, culturing the third TIL population is performed for about 1 to 7 days. In some embodiments, culturing the third TIL population is performed for about 1 to 7 days, about 2 to 7 days, about 3 to 7 days, about 4 to 7 days, about 5 to 7 days, about 6 to 7 days, about 1 to 6 days, about 2 to 6 days, about 3 to 6 days, about 4 to 6 days, about 5 to 6 days, about 1 to 5 days, about 2 to 5 days, about 3 to 5 days, about 4 to 5 days, about 1 to 4 days, about 2 to 4 days, about 3 to 4 days, about 1 to 3 days, about 2 to 3 days, or about 1 to 2 days. In some embodiments, culturing the third TIL population is performed for about 1 day. In some embodiments, culturing the third TIL population is performed for about 2 days. In some embodiments, culturing the third TIL population is performed for about 3 days. In some embodiments, culturing the third TIL population is performed for about 4 days. In some embodiments, culturing the third TIL population is performed for about 5 days. In some embodiments, culturing the third TIL population is performed for about 6 days. In some embodiments, culturing the third TIL population is performed for about 7 days.

[0607] In some embodiments, culturing each of the plurality of subcultures is carried out for about 3-6 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 3-6 days, about 4-6 days, about 5-6 days, about 3-5 days, about 4-5 days, or about 3-4 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 3 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 4 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 5 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 6 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 7 days.

[0608] In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 8 days. In some embodiments, the steps of this method are completed within a time period of about 9 days. In some embodiments, the steps of this method are completed within a time period of about 10 days. In some embodiments, the steps of this method are completed within a time period of about 11 days. In some embodiments, the steps of this method are completed within a time period of about 12 days. In some embodiments, the steps of this method are completed within a time period of about 13 days. In some embodiments, the steps of this method are completed within a time period of about 14 days. In some embodiments, the steps of this method are completed within a time period of about 15 days. In some embodiments, the steps of this method are completed within a time period of about 16 days. In some embodiments, the steps of this method are completed within a time period of about 17 days. In some embodiments, the steps of this method are completed within a time period of about 18 days. In some embodiments, the steps of this method are completed within a time period of about 19 days. In some embodiments, the steps of this method are completed within a time period of about 20 days. In some embodiments, the steps of this method are completed within a period of about 21 days. In some embodiments, the steps of this method are completed within a period of about 22 days. In some embodiments, the steps of this method are completed within a period of about 23 days.

[0609] In some embodiments, the gene editing process can be performed at any time during the TIL expansion method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, between any of steps (a)-(e) or (a)-(f) outlined in the method above, or before or after any of steps (a)-(e) or (a)-(f) outlined in the method above. In some embodiments, the gene editing process can be performed more than once at any time during the TIL expansion method. According to certain embodiments, the TILs are collected during the culture step (e.g., the culture step is "paused" for at least some of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the culture step (e.g., returned to culture medium) to continue the culture step, thereby permanently gene editing at least some of the therapeutic TIL population that will ultimately be transferred to the infusion bag.

[0610] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific embodiment, the gene editing process may be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two culture steps, and gene editing may be performed on the TIL during the third or fourth culture step, etc.

[0611] According to some embodiments, gene editing is performed while the TILs are still in the culture medium and while the culturing step is being performed, i.e., they are not necessarily "removed" from the culture step to perform the gene editing. According to some embodiments, gene editing is performed on TILs that have been harvested from the culture medium, and after the gene editing process, the TILs are then returned to the culture medium.

[0612] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population; (c) culturing the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) culturing the fourth TIL population in a third cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0613] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population; (d) culturing the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to produce a third TIL population; (e) gene editing at least a portion of the third TIL population to produce a fourth TIL population; (f) culturing the fourth TIL population in a third cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

[0614] In some embodiments, culturing the second TIL population is performed for about 2-4 days. In some embodiments, culturing the third TIL population is performed for about 2-4 days, about 3-4 days, or about 2-3 days. In some embodiments, culturing the second TIL population is performed for about 2 days. In some embodiments, culturing the second TIL population is performed for about 3 days. In some embodiments, culturing the second TIL population is performed for about 4 days.

[0615] In some embodiments, culturing the fourth TIL population is carried out for about 5 to 15 days. In some embodiments, culturing the fourth TIL population is carried out for about 5 to 15 days, about 6 to 15 days, about 7 to 15 days, about 8 to 15 days, about 9 to 15 days, about 10 to 15 days, about 11 to 15 days, about 12 to 15 days, about 13 to 15 days, about 14 to 15 days, about 5 to 14 days, about 6 to 14 days, about 7 to 14 days, about 8 to 14 days, about 9 to 14 days, about 10 to 14 days, about 11 to 14 days, about 12 to 14 days, about 13 to 14 days, about 5 to 13 days, about 6 to 13 days, about 7 to 13 days, about 8 to 13 days, about 9 to 13 days, about 10 to 15 days. In some embodiments, culturing the fourth TIL population is performed for about 13 days, about 11-13 days, about 12-13 days, about 5-12 days, about 6-12 days, about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, about 11-12 days, about 5-11 days, 6-11 days, 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 5-10 days, 6-10 days, 7-10 days, about 8-10 days, about 9-10 days, about 5-9 days, 6-9 days, 7-9 days, about 8-9 days, about 5-8 days, about 6-8 days, 7-8 days, about 5-7 days, about 6-7 days, or about 5-6 days. In some embodiments, culturing the fourth TIL population is performed for about 5 days. In some embodiments, culturing the fourth TIL population is performed for about 6 days. In some embodiments, culturing the fourth TIL population is performed for about 7 days. In some embodiments, culturing the fourth TIL population is performed for about 8 days. In some embodiments, culturing the fourth TIL population is performed for about 9 days. In some embodiments, culturing the fourth TIL population is performed for about 10 days. In some embodiments, culturing the fourth TIL population is performed for about 11 days. In some embodiments, culturing the fourth TIL population is performed for about 12 days. In some embodiments, culturing the fourth TIL population is performed for about 13 days. In some embodiments, culturing the fourth TIL population is performed for about 14 days. In some embodiments, culturing the fourth TIL population is performed for about 15 days.

[0616] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0617] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0618] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0619] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0620] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0621] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0622] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0623] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0624] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0625] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0626] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0627] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0628] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0629] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0630] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0631] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0632] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0633] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0634] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0635] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0636] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0637] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0638] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0639] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0640] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0641] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0642] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0643] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0644] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0645] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0646] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0647] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0648] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0649] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0650] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0651] In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 8 days. In some embodiments, the steps of this method are completed within a time period of about 9 days. In some embodiments, the steps of this method are completed within a time period of about 10 days. In some embodiments, the steps of this method are completed within a time period of about 11 days. In some embodiments, the steps of this method are completed within a time period of about 12 days. In some embodiments, the steps of this method are completed within a time period of about 13 days. In some embodiments, the steps of this method are completed within a time period of about 14 days. In some embodiments, the steps of this method are completed within a time period of about 15 days. In some embodiments, the steps of this method are completed within a time period of about 16 days. In some embodiments, the steps of this method are completed within a time period of about 17 days. In some embodiments, the steps of this method are completed within a time period of about 18 days. In some embodiments, the steps of this method are completed within a time period of about 19 days. In some embodiments, the steps of this method are completed within a time period of about 20 days. In some embodiments, the steps of this method are completed within a period of about 21 days. In some embodiments, the steps of this method are completed within a period of about 22 days.

[0652] In some embodiments, the gene editing process may be performed at any time during the TIL expansion method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, between any of steps (a)-(f) or (a)-(g) outlined in the method above, or before or after any of steps (a)-(f) or (a)-(g) outlined in the method above. In some embodiments, the gene editing process may be performed more than once at any time during the TIL expansion method. According to certain embodiments, the TILs are collected during the culture step (e.g., the culture step is "paused" for at least some of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the culture step (e.g., returned to culture medium) to continue the culture step, thereby permanently gene editing at least some of the therapeutic TIL population that will ultimately be transferred to the infusion bag.

[0653] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific embodiment, the gene editing process may be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two culture steps, and gene editing may be performed on the TIL during the third or fourth culture step, etc.

[0654] According to some embodiments, gene editing is performed while the TILs are still in the culture medium and while the culturing step is being performed, i.e., they are not necessarily "removed" from the culture step to perform the gene editing. According to some embodiments, gene editing is performed on TILs that have been harvested from the culture medium, and after the gene editing process, the TILs are then returned to the culture medium.

[0655] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population; (c) culturing the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) culturing the third TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 1 to 7 days to produce a culture of a fourth TIL population; (f) dividing the culture of the fourth TIL population into a plurality of subcultures, culturing each of the plurality of subcultures in a third cell culture medium containing IL-2 for about 3 to 7 days, and combining the plurality of subcultures to provide a fifth TIL population comprising an expanded number of TILs.

[0656] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor tissue in an enzymatic medium to produce a tumor digest; (c) culturing the first TIL population in a first cell culture medium containing IL-2 for about 3 days to produce a second TIL population; (d) culturing the second TIL population in a second cell culture medium containing IL-2 and OKT-3 for 2-4 days to produce a third TIL population; (e) gene editing at least a portion of the third TIL population to produce a fourth TIL population; (f) culturing the fourth TIL population in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 1 to 7 days to produce a culture of a fifth TIL population; (g) dividing the culture of the fifth TIL population into a plurality of subcultures, culturing each of the plurality of subcultures in a third cell culture medium containing IL-2 for about 3 to 7 days, and combining the plurality of subcultures to provide a fifth TIL population comprising an expanded number of TILs.

[0657] In some embodiments, culturing the second TIL population is performed for about 2-4 days. In some embodiments, culturing the third TIL population is performed for about 2-4 days, about 3-4 days, or about 2-3 days. In some embodiments, culturing the second TIL population is performed for about 2 days. In some embodiments, culturing the second TIL population is performed for about 3 days. In some embodiments, culturing the second TIL population is performed for about 4 days.

[0658] In some embodiments, culturing the fourth TIL population is performed for about 1-7 days. In some embodiments, culturing the fourth TIL population is performed for about 1-7 days, about 1-6 days, about 2-6 days, about 3-6 days, about 4-6 days, about 5-6 days, about 1-5 days, about 2-5 days, about 3-5 days, about 4-5 days, about 1-4 days, about 2-4 days, about 3-4 days, about 1-3 days, about 2-3 days, or about 1-2 days. In some embodiments, culturing the fourth TIL population is performed for about 1 day. In some embodiments, culturing the fourth TIL population is performed for about 2 days. In some embodiments, culturing the fourth TIL population is performed for about 3 days. In some embodiments, culturing the fourth TIL population is performed for about 4 days. In some embodiments, culturing the fourth TIL population is performed for about 5 days. In some embodiments, culturing the fourth population of TILs is performed for about 6 days. In some embodiments, culturing the fourth population of TILs is performed for about 7 days.

[0659] In some embodiments, culturing each of the plurality of subcultures is carried out for about 3-6 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 3-6 days, about 4-6 days, about 5-6 days, about 3-5 days, about 4-5 days, or about 3-4 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 3 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 4 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 5 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 6 days. In some embodiments, culturing each of the plurality of subcultures is carried out for about 7 days.

[0660] In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 8 days. In some embodiments, the steps of this method are completed within a time period of about 9 days. In some embodiments, the steps of this method are completed within a time period of about 10 days. In some embodiments, the steps of this method are completed within a time period of about 11 days. In some embodiments, the steps of this method are completed within a time period of about 12 days. In some embodiments, the steps of this method are completed within a time period of about 13 days. In some embodiments, the steps of this method are completed within a time period of about 14 days. In some embodiments, the steps of this method are completed within a time period of about 15 days. In some embodiments, the steps of this method are completed within a time period of about 16 days. In some embodiments, the steps of this method are completed within a time period of about 17 days. In some embodiments, the steps of this method are completed within a time period of about 18 days. In some embodiments, the steps of this method are completed within a time period of about 19 days. In some embodiments, the steps of this method are completed within a time period of about 20 days. In some embodiments, the steps of this method are completed within a period of about 21 days.

[0661] In some embodiments, the gene editing process may be performed at any time during the TIL expansion method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, between any of steps (a)-(f) or (a)-(g) outlined in the method above, or before or after any of steps (a)-(f) or (a)-(g) outlined in the method above. In some embodiments, the gene editing process may be performed more than once at any time during the TIL expansion method. According to certain embodiments, the TILs are collected during the culture step (e.g., the culture step is "paused" for at least some of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the culture step (e.g., returned to culture medium) to continue the culture step, thereby permanently gene editing at least some of the therapeutic TIL population that will ultimately be transferred to the infusion bag.

[0662] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific embodiment, the gene editing process may be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two culture steps, and gene editing may be performed on the TIL during the third or fourth culture step, etc.

[0663] According to some embodiments, gene editing is performed while the TILs are still in the culture medium and while the culturing step is being performed, i.e., they are not necessarily "removed" from the culture step to perform the gene editing. According to some embodiments, gene editing is performed on TILs that have been harvested from the culture medium, and after the gene editing process, the TILs are then returned to the culture medium.

[0664] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 3 to 9 days; (c) activating the second TIL population for 1 to 7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) performing a second rapid expansion of the fourth TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3, and APC, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0665] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 3 to 9 days; (c) gene editing at least a portion of the second TIL population to produce a third TIL population; (d) performing a rapid second expansion of the third TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3, and APC, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0666] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 9 days; (d) activating the second TIL population for 1 to 7 days using anti-CD3 and anti-CD28 beads or antibodies to produce a third TIL population; (e) gene editing at least a portion of the third TIL population to produce a fourth TIL population; (f) performing a rapid second expansion of the fourth TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3, and APC, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0667] In some embodiments, the method for preparing expanded tumor infiltrating lymphocytes (TILs) comprises: (a) obtaining and / or receiving a first population of TILs from tumor tissue resected from a subject or patient; (b) digesting the tumor fragments in an enzymatic medium to produce a tumor digest; (c) performing an initial expansion (or first expansion by priming) of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of about 1 to 9 days; (d) gene editing at least a portion of the second TIL population to produce a third TIL population; (e) performing a rapid second expansion of the third TIL population in a second cell culture medium to obtain an expanded number of TILs, wherein the second cell culture medium comprises IL-2, OKT-3, and APC, and the rapid expansion is performed over a period of 14 days or less, and optionally, the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the initiation of the rapid second expansion to obtain an expanded number of TILs.

[0668] In some embodiments, the initial expansion is carried out for about 3 to 9 days. In some embodiments, the initial expansion is carried out for about 1 to 9 days, 2 to 9 days, 3 to 9 days, about 4 to 9 days, about 5 to 9 days, about 6 to 9 days, about 7 to 9 days, about 8 to 9 days, about 1 to 8 days, about 2 to 8 days, about 3 to 8 days, about 4 to 8 days, about 5 to 8 days, about 6 to 8 days, about 7 to 8 days, about 1 to 7 days, about 2 to 7 days, about 3 to 7 days, about 4 to 7 days, about 5 to 7 days, about 6 to 7 days, about 1 to 6 days, about 2 to 6 days, about 3 to 6 days, about 4 to 6 days, about 5 to 6 days, about 1 to 5 days, about 2 to 5 days, about 3 to 5 days, about 4 to 5 days, about 1 to 4 days, about 2 to 4 days, about 3 to 4 days, about 1 to 3 days, about 2 to 3 days, or about 1 to 2 days. In some embodiments, the initial expansion occurs for about 1 day. In some embodiments, the initial expansion occurs for about 2 days. In some embodiments, the initial expansion occurs for about 3 days. In some embodiments, the initial expansion occurs for about 4 days. In some embodiments, the initial expansion occurs for about 5 days. In some embodiments, the initial expansion occurs for about 6 days. In some embodiments, the initial expansion occurs for about 7 days. In some embodiments, the initial expansion occurs for about 8 days. In some embodiments, the initial expansion occurs for about 9 days.

[0669] In some embodiments, the step of activating the second TIL population is carried out for about 1 to 7 days. In some embodiments, the step of activating the second TIL population is carried out for about 1 to 7 days, about 2 to 7 days, about 3 to 7 days, about 4 to 7 days, about 5 to 7 days, about 6 to 7 days, about 1 to 6 days, about 2 to 6 days, about 3 to 6 days, about 4 to 6 days, about 5 to 6 days, about 1 to 5 days, about 2 to 5 days, about 3 to 5 days, about 4 to 5 days, about 1 to 4 days, about 2 to 4 days, about 3 to 4 days, about 1 to 3 days, about 2 to 3 days, or about 1 to 2 days. In some embodiments, the step of activating the second TIL population is carried out for about 1 day. In some embodiments, the step of activating the second TIL population is carried out for about 2 days. In some embodiments, the step of activating the second TIL population is carried out for about 3 days. In some embodiments, the step of activating the second TIL population is carried out for about 4 days. In some embodiments, the step of activating the second TIL population is carried out for about 5 days. In some embodiments, the step of activating the second TIL population is carried out for about 6 days. In some embodiments, the step of activating the second TIL population is carried out for about 7 days.

[0670] In some embodiments, the rapid second expansion is carried out for about 5 to 15 days. In some embodiments, the rapid second expansion is carried out for about 5 to 15 days, about 6 to 15 days, about 7 to 15 days, about 8 to 15 days, about 9 to 15 days, about 10 to 15 days, about 11 to 15 days, about 12 to 15 days, about 13 to 15 days, about 14 to 15 days, about 5 to 14 days, about 6 to 14 days, about 7 to 14 days, about 8 to 14 days, about 9 to 14 days, about 10 to 14 days, about 11 to 14 days, about 12 to 14 days, about 13 to 14 days, about 5 to 13 days, about 6 to 13 days, about 7 to 13 days, about 8 to 13 days, about 9 to 13 days, about 10 to 13 days, The rapid second expansion is performed for about 11-13 days, about 12-13 days, about 5-12 days, about 6-12 days, about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, about 11-12 days, about 5-11 days, 6-11 days, 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 5-10 days, 6-10 days, 7-10 days, about 8-10 days, about 9-10 days, about 5-9 days, 6-9 days, 7-9 days, about 8-9 days, about 5-8 days, about 6-8 days, 7-8 days, about 5-7 days, about 6-7 days, or about 5-6 days. In some embodiments, the rapid second expansion is performed for about 5 days. In some embodiments, the rapid second expansion is performed for about 6 days. In some embodiments, the rapid second expansion is performed for about 7 days. In some embodiments, the rapid second expansion occurs for about 8 days. In some embodiments, the rapid second expansion occurs for about 9 days. In some embodiments, the rapid second expansion occurs for about 10 days. In some embodiments, the rapid second expansion occurs for about 11 days. In some embodiments, the rapid second expansion occurs for about 12 days. In some embodiments, the rapid second expansion occurs for about 13 days. In some embodiments, the rapid second expansion occurs for about 14 days. In some embodiments, the rapid second expansion occurs for about 15 days.

[0671] In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 8 days. In some embodiments, the steps of this method are completed within a time period of about 9 days. In some embodiments, the steps of this method are completed within a time period of about 10 days. In some embodiments, the steps of this method are completed within a time period of about 11 days. In some embodiments, the steps of this method are completed within a time period of about 12 days. In some embodiments, the steps of this method are completed within a time period of about 13 days. In some embodiments, the steps of this method are completed within a time period of about 14 days. In some embodiments, the steps of this method are completed within a time period of about 15 days. In some embodiments, the steps of this method are completed within a time period of about 16 days. In some embodiments, the steps of this method are completed within a time period of about 17 days. In some embodiments, the steps of this method are completed within a time period of about 18 days. In some embodiments, the steps of this method are completed within a time period of about 19 days. In some embodiments, the steps of this method are completed within a time period of about 20 days. In some embodiments, the steps of this method are completed within a time period of about 21 days. In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 23 days. In some embodiments, the steps of this method are completed within a time period of about 24 days. In some embodiments, the steps of this method are completed within a time period of about 25 days. In some embodiments, the steps of this method are completed within a time period of about 26 days. In some embodiments, the steps of this method are completed within a time period of about 27 days. In some embodiments, the steps of this method are completed within a time period of about 28 days. In some embodiments, the steps of this method are completed within a time period of about 29 days. In some embodiments, the steps of this method are completed within a time period of about 30 days. In some embodiments, the steps of this method are completed within a time period of about 31 days.

[0672] In some embodiments, the rapid second expansion is carried out by culturing the third TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0673] In some embodiments, the rapid second expansion is carried out by culturing the third TIL population in a second culture medium for a first period of about 5 days, at the end of the first period the culture is divided into multiple subcultures, each of the multiple subcultures is cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period the multiple subcultures are combined to provide an expanded number of TILs.

[0674] In some embodiments, the gene editing process can be performed at any time during the TIL expansion method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, between any of steps (a)-(e) or (a)-(f) outlined in the method above, or before or after any of steps (a)-(e) or (a)-(f) outlined in the method above. In some embodiments, the gene editing process can be performed more than once at any time during the TIL expansion method. According to certain embodiments, the TILs are collected during the culture step (e.g., the culture step is "paused" for at least some of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the culture step (e.g., returned to culture medium) to continue the culture step, thereby permanently gene editing at least some of the therapeutic TIL population that will ultimately be transferred to the infusion bag.

[0675] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(e) or (a)-(f), or may have a different number of steps. Regardless of the specific embodiment, the gene editing process may be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two culture steps, and gene editing may be performed on the TIL during the third or fourth culture step, etc.

[0676] According to some embodiments, gene editing is performed while the TILs are still in the culture medium and while the culturing step is being performed, i.e., they are not necessarily "removed" from the culture step to perform the gene editing. According to some embodiments, gene editing is performed on TILs that have been harvested from the culture medium, and after the gene editing process, the TILs are then returned to the culture medium.

[0677] In some embodiments, the method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population comprises: (a) obtaining and / or receiving a first population of TILs from a tumor tissue sample made by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject; (b) adding the tumor tissue to a closed system and performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 9 days to obtain the second TIL population; (c) activating the second TIL population using CD3 and CD28 beads or antibodies for 1 to 7 days to produce a third TIL population; (d) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (e) performing a second expansion of the fourth TIL population by culturing it in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a fifth TIL population, wherein the second expansion is performed for about 5-15 days to obtain a third TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, wherein the fifth TIL population is a therapeutic TIL population; (f) harvesting the therapeutic TIL population obtained from step (e), wherein each of steps (b) through (f) is performed in a closed sterile system, and the transition from step (b) to step (c), from step (c) to step (d), from step (d) to step (e), and / or the transition from step (e) to step (f) occurs without opening the system.

[0678] In some embodiments, the method for expanding tumor-infiltrating lymphocytes into a therapeutic TIL population comprises: (a) obtaining and / or receiving a first population of TILs from a tumor tissue sample made by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining tumor tissue from a patient or subject; (b) digesting a sample of tumor tissue or tumor fragment in an enzymatic medium to produce a tumor digest; (c) adding the tumor tissue to a closed system and performing a first expansion by culturing the first TIL population in a first cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 9 days to obtain the second TIL population; (d) activating the second TIL population using CD3 and CD28 beads or antibodies for 1 to 7 days to produce a third TIL population; (e) gene editing at least a portion of the third TIL population to produce a fourth TIL population; (f) performing a second expansion of the fourth TIL population by culturing it in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a fifth TIL population, wherein the second expansion is performed for about 5-15 days to obtain a third TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, wherein the fifth TIL population is a therapeutic TIL population; (g) harvesting the therapeutic TIL population obtained from step (e), wherein each of steps (c) through (g) is performed in a closed sterile system, and the transition from step (c) to step (d), from step (d) to step (e), from step (e) to step (f), and / or from step (f) to step (g) occurs without opening the system.

[0679] In some embodiments, the first expansion is performed for about 3 to 9 days. In some embodiments, the first expansion is performed for about 3 to 9 days, about 3 to 8 days, about 3 to 7 days, about 3 to 6 days, about 3 to 5 days, about 3 to 4 days, about 4 to 9 days, about 4 to 8 days, about 5 to 9 days, about 5 to 8 days, about 6 to 9 days, about 6 to 8 days, about 7 to 9 days, about 7 to 8 days, about 3 to 7 days, about 4 to 7 days, about 5 to 7 days, about 6 to 7 days, about 3 to 6 days, about 4 to 6 days, about 5 to 6 days, about 3 to 5 days, about 4 to 5 days, or about 3 to 4 days. In some embodiments, the first expansion is performed for about 3 days. In some embodiments, the first expansion is performed for about 4 days. In some embodiments, the first expansion is performed for about 5 days. In some embodiments, the first expansion is performed for about 6 days. In some embodiments, the first expansion is performed for about 7 days. In some embodiments, the first expansion is performed for about 8 days. In some embodiments, the first expansion is performed for about 9 days.

[0680] In some embodiments, the step of activating the second TIL population is carried out for about 1 to 7 days. In some embodiments, the step of activating the second TIL population is carried out for about 1 to 7 days, about 2 to 7 days, about 3 to 7 days, 4 to 7 days, about 5 to 7 days, about 6 to 7 days, about 1 to 6 days, about 2 to 6 days, about 3 to 6 days, about 4 to 6 days, about 5 to 6 days, about 1 to 5 days, about 2 to 5 days, about 3 to 5 days, about 4 to 5 days, about 1 to 4 days, about 2 to 4 days, about 3 to 4 days, about 1 to 3 days, about 2 to 3 days, or about 1 to 2 days. In some embodiments, the step of activating the second TIL population is carried out for about 1 day. In some embodiments, the step of activating the second TIL population is carried out for about 2 days. In some embodiments, the step of activating the second TIL population is carried out for about 3 days. In some embodiments, the step of activating the second TIL population is carried out for about 4 days. In some embodiments, the step of activating the second TIL population is carried out for about 5 days. In some embodiments, the step of activating the second TIL population is carried out for about 6 days. In some embodiments, the step of activating the second TIL population is carried out for about 7 days.

[0681] In some embodiments, the second expansion is carried out for about 5-15 days. In some embodiments, the second expansion is carried out for about 5-15 days, about 6-15 days, about 7-15 days, about 8-15 days, about 9-15 days, about 10-15 days, about 11-15 days, about 12-15 days, about 13-15 days, about 14-15 days, about 5-14 days, about 6-14 days, about 7-14 days, about 8-14 days, about 9-14 days, about 10-14 days, about 11-14 days, about 12-14 days, about 13-14 days, about 5-13 days, about 6-13 days, about 7-13 days, about 8-13 days, about 9-13 days, about 10-13 days, or about The second expansion is performed for 11-13 days, about 12-13 days, about 5-12 days, about 6-12 days, about 7-12 days, about 8-12 days, about 9-12 days, about 10-12 days, about 11-12 days, about 5-11 days, 6-11 days, 7-11 days, about 8-11 days, about 9-11 days, about 10-11 days, about 5-10 days, 6-10 days, 7-10 days, about 8-10 days, about 9-10 days, about 5-9 days, 6-9 days, 7-9 days, about 8-9 days, about 5-8 days, about 6-8 days, 7-8 days, about 5-7 days, about 6-7 days, or about 5-6 days. In some embodiments, the second expansion is performed for about 5 days. In some embodiments, the second expansion is performed for about 6 days. In some embodiments, the second expansion is performed for about 7 days. In some embodiments, the second expansion is performed for about 8 days. In some embodiments, the second expansion is performed for about 9 days. In some embodiments, the second expansion is performed for about 10 days. In some embodiments, the second expansion is performed for about 11 days. In some embodiments, the second expansion is performed for about 12 days. In some embodiments, the second expansion is performed for about 13 days. In some embodiments, the second expansion is performed for about 14 days. In some embodiments, the second expansion is performed for about 15 days.

[0682] In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 8 days. In some embodiments, the steps of this method are completed within a time period of about 9 days. In some embodiments, the steps of this method are completed within a time period of about 10 days. In some embodiments, the steps of this method are completed within a time period of about 11 days. In some embodiments, the steps of this method are completed within a time period of about 12 days. In some embodiments, the steps of this method are completed within a time period of about 13 days. In some embodiments, the steps of this method are completed within a time period of about 14 days. In some embodiments, the steps of this method are completed within a time period of about 15 days. In some embodiments, the steps of this method are completed within a time period of about 16 days. In some embodiments, the steps of this method are completed within a time period of about 17 days. In some embodiments, the steps of this method are completed within a time period of about 18 days. In some embodiments, the steps of this method are completed within a time period of about 19 days. In some embodiments, the steps of this method are completed within a time period of about 20 days. In some embodiments, the steps of this method are completed within a time period of about 21 days. In some embodiments, the steps of this method are completed within a time period of about 22 days. In some embodiments, the steps of this method are completed within a time period of about 23 days. In some embodiments, the steps of this method are completed within a time period of about 24 days. In some embodiments, the steps of this method are completed within a time period of about 25 days. In some embodiments, the steps of this method are completed within a time period of about 26 days. In some embodiments, the steps of this method are completed within a time period of about 27 days. In some embodiments, the steps of this method are completed within a time period of about 28 days. In some embodiments, the steps of this method are completed within a time period of about 29 days. In some embodiments, the steps of this method are completed within a time period of about 30 days. In some embodiments, the steps of this method are completed within a time period of about 31 days. In some embodiments, the steps of this method are completed within a time period of about 32 days.

[0683] In some embodiments, the second expansion is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0684] In some embodiments, the second expansion is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0685] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0686] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0687] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0688] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0689] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 1 day, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0690] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0691] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0692] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0693] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0694] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 2 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0695] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0696] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0697] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 5 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0698] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0699] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 3 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0700] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0701] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0702] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0703] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0704] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 4 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0705] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0706] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0707] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0708] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 6 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0709] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 5 days, at the end of the first period, dividing the culture into multiple subcultures, and culturing each of the multiple subcultures in a third culture medium containing IL-2 for a second period of about 7 days, at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0710] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0711] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0712] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0713] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0714] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 6 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0715] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 3 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0716] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 4 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0717] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 5 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0718] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 6 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0719] In some embodiments, the step of culturing the fourth TIL population is carried out by culturing the fourth TIL population in a second culture medium for a first period of about 7 days, at the end of the first period, dividing the culture into multiple subcultures, each of the multiple subcultures being cultured in a third culture medium containing IL-2 for a second period of about 7 days, and at the end of the second period, combining the multiple subcultures to provide an expanded number of TILs.

[0720] In some embodiments, the gene editing process may be performed at any time during the TIL expansion method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps of the expansion method, for example, between any of steps (a)-(f) or (a)-(g) outlined in the method above, or before or after any of steps (a)-(f) or (a)-(g) outlined in the method above. In some embodiments, the gene editing process may be performed more than once at any time during the TIL expansion method. According to certain embodiments, the TILs are collected during the culture step (e.g., the culture step is "paused" for at least some of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the culture step (e.g., returned to culture medium) to continue the culture step, thereby permanently gene editing at least some of the therapeutic TIL population that will ultimately be transferred to the infusion bag.

[0721] It should be noted that alternative embodiments of the expansion process may differ from the methods shown above; for example, alternative embodiments may not have the same steps (a)-(f) or (a)-(g), or may have a different number of steps. Regardless of the specific embodiment, the gene editing process may be performed at any time during the TIL expansion method. For example, alternative embodiments may include more than two culture steps, and gene editing may be performed on the TIL during the third or fourth culture step, etc.

[0722] In some embodiments, gene editing is performed while the TILs are still in the culture medium and while the culturing step is being performed, i.e., they are not necessarily "removed" from the culture step to perform the gene editing. According to some embodiments, gene editing is performed on TILs that have been harvested from the culture medium, and after the gene editing process, the TILs are then returned to the culture medium.

[0723] In some embodiments, the step of gene editing at least a portion of the second or third TIL population comprises performing a sterile electroporation step on the second or third TIL population.

[0724] In some embodiments, the sterile electroporation step mediates the transfer of at least one gene editor. According to some embodiments, the gene editor is a TALE-nuclease system for modulating expression of at least one protein. According to some embodiments, the TALE-nuclease system downregulates expression of PD-1. According to some embodiments, the gene editor further comprises a TALE-nuclease system that downregulates expression of CTLA-4. According to some embodiments, the gene editor further comprises a TALE-nuclease system that downregulates expression of LAG-3. According to some embodiments, the gene editor further comprises a TALE-nuclease system that downregulates expression of CISH. According to some embodiments, the gene editor further comprises a TALE-nuclease system that downregulates expression of CBL-B. According to some embodiments, the gene editor further comprises a TALE-nuclease system that downregulates expression of TIGIT. According to some embodiments, the resulting TILs are PD-1 knockout TILs. According to some embodiments, the resulting TILs are CTLA-4 knockout TILs. According to some embodiments, the resulting TILs are LAG-3 knockout TILs. According to some embodiments, the resulting TILs are CISH knockout TILs. According to some embodiments, the resulting TILs are CBL-B knockout TILs. According to some embodiments, the resulting TILs are TIGIT knockout TILs. According to some embodiments, the resulting TILs exhibit downregulated expression of PD-1 and downregulated expression of one or more of CTLA-4, LAG-3, CISH, TIGIT, and CBL-B. According to some embodiments, the resulting TILs exhibit downregulated expression of CTLA-4 and downregulated expression of one or more of PD-1, LAG-3, CISH, TIGIT, and CBL-B. According to some embodiments, the resulting TILs exhibit downregulated expression of LAG-3 and downregulated expression of one or more of PD-1, CTLA-4, CISH, TIGIT, and CBL-B.According to some embodiments, the resulting TILs exhibit downregulated expression of CISH and downregulated expression of one or more of PD-1, LAG-3, CTLA-4, TIGIT, and CBL-B. According to some embodiments, the resulting TILs exhibit downregulated expression of CBL-B and downregulated expression of one or more of CTLA-4, LAG-3, CISH, TIGIT, and PD-1. According to some embodiments, the resulting TILs are PD-1 / CTLA-4 double knockout TILs. According to some embodiments, the resulting TILs are PD-1 / LAG-3 double knockout TILs. According to some embodiments, the resulting TILs are PD-1 / CISH double knockout TILs. According to some embodiments, the resulting TILs are PD-1 / CBL-B double knockout TILs. According to some embodiments, the resulting TILs are PD-1 / TIGIT double knockout TILs. According to some embodiments, the resulting TILs are CTLA-4 / LAG-3 double knockout TILs. According to some embodiments, the resulting TILs are CTLA-4 / CISH double knockout TILs. According to some embodiments, the resulting TILs are CTLA-4 / CBL-B double knockout TILs. According to some embodiments, the resulting TILs are CTLA-4 / TIGIT double knockout TILs. According to some embodiments, the resulting TILs are LAG-3 / CISH double knockout TILs. According to some embodiments, the resulting TILs are LAG-3 / CBL-B double knockout TILs. According to some embodiments, the resulting TILs are LAG-3 / TIGIT double knockout TILs. According to some embodiments, the resulting TILs are CISH / CBL-B double knockout TILs. According to some embodiments, the resulting TILs are CISH / TIGIT double knockout TILs. According to some embodiments, the resulting TILs are CBL-B / TIGIT double knockout TILs.

[0725] In some embodiments, the gene editing step further comprises a incubation step. According to some embodiments, the incubation step comprises incubating the fourth TIL population at about 30-40° C. and about 5% CO2. According to some embodiments, the incubation step is performed at about 30° C., about 30.5° C., about 31° C., about 31.5° C., about 32° C., about 32.5° C., about 33° C., about 33.5° C., about 34° C., about 34.5° C., about 35° C., about 35.5° C., about 36° C., about 36.5° C., about 37° C., about 37.5° C., about 38° C., about 38.5° C., about 39° C., about 39.5° C., or about 40° C. According to some embodiments, the incubation step is carried out for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. According to some embodiments, the incubation step comprises incubating the third or fourth TIL population in ...

Claims

1. 1. A method for preparing expanded tumor infiltrating lymphocytes (TILs), comprising: (a) culturing a first population of TILs obtained and / or received from tumor tissue resected from a subject or patient in a first cell culture medium containing IL-2 for about 3-12 days to produce a second population of TILs; (b) activating the second TIL population using anti-CD3 agonist beads or antibodies, or anti-CD3 agonist and anti-CD28 agonist beads or antibodies, for 1 to 7 days to produce a third TIL population; (c) gene editing at least a portion of the third TIL population to produce a fourth TIL population; and (d) culturing the fourth population of TILs in a second cell culture medium comprising antigen-presenting cells (APCs), OKT-3, and IL-2 for about 5 to 15 days to produce an expanded number of TILs.

2. 10. The method of claim 1, wherein the first culture period is about 11 days.

3. 3. The method of claim 1 or 2, wherein the second culture period is about 11 days.

4. 3. The method of claim 1 or 2, wherein the step of activating the second TIL population is performed using anti-CD3 agonist and anti-CD28 agonist beads or antibodies.

5. 5. The method of claim 4, wherein the step of activating the second TIL population is performed using TransAct.

6. 6. The method of claim 5, wherein the step of activating the second TIL population is performed using TransAct at a dilution of 1:10, 1:17.5, or 1:

100.

7. The method of claim 1 or 2, wherein the expanded number of TILs comprises a therapeutic TIL population.

8. 3. The method of Claim 1 or 2, wherein the step of gene editing at least a portion of the third TIL population comprises performing a sterile electroporation step on the third TIL population, wherein the sterile electroporation step mediates the transfer of at least one gene editor.

9. 10. The method of claim 8, wherein the at least one gene editor is a TALE nuclease system for regulating expression of at least one protein.

10. 9. The method of claim 8, wherein the at least one gene editor comprises a TALE nuclease system that regulates expression of PD-1, CTLA-4, LAG-3, CISH, CBL-B, and / or TIGIT.

11. The method of claim 8, wherein the sterile electroporation step mediates the introduction of at least two gene editors, the at least two gene editors comprising a first gene editor comprising a first TALE nuclease system for regulating expression of a first protein, and a second gene editor comprising a second TALE nuclease system for regulating expression of a second protein.

12. 12. The method of claim 11, wherein the first and second TALE nuclease systems regulate expression of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and / or CBL-B.

13. 12. The method of claim 11, wherein the first protein and the second protein are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein and the second protein are different.

14. 12. The method of Claim 11, wherein the first gene editor downregulates expression of the first protein and the second gene editor downregulates expression of the second protein.

15. 1. A gene-edited population of tumor-infiltrating lymphocytes (TILs), comprising an expanded TIL population, wherein expression of at least one protein is regulated by a gene editor transferred into at least a portion of the expanded TIL population.

16. 16. The gene-edited TIL population of Claim 15, wherein the gene editor is a TALE nuclease system for regulating the expression of the at least one protein.

17. 17. The gene-edited TIL population of Claim 15 or 16, wherein expression of at least two proteins is regulated by at least two gene editors transferred into at least a portion of the expanded TIL population, wherein the at least two gene editors comprise a first gene editor comprising a first TALE nuclease system for regulating expression of a first protein, and a second gene editor comprising a second TALE nuclease system for regulating expression of a second protein.

18. 18. The gene-edited TIL population of claim 17, wherein the first and second proteins are independently selected from the group consisting of PD-1, CTLA-4, LAG-3, CISH, TIGIT, and CBL-B, provided that the first protein and the second protein are different.

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