A method for tumor-infiltrating lymphocyte (TIL) expansion in conjunction with CD39 / CD103 selection

Abstract

To provide an improved and / or shortened process and method for preparing TILs with specific combinations of cytokines and other cell culture medium components to prepare therapeutic TIL populations with increased therapeutic efficacy for the treatment of cancer with TILs. [Solution] Provided herein are TILs that are CD39, CD103, and / or both. In some embodiments, the subject TILs are produced by genetically engineering a population of TILs selected for expression of (i) PD-1, (ii) CD39, (iii) CD103, (iv) a combination of (i) and (ii), (v) a combination of (i) and (iii), or (vi) a combination of (ii) and (iii). Expansion methods for producing such TIL populations and therapeutic methods using such TILs are also provided herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 382,460, filed November 4, 2022, the entire disclosure of which is incorporated herein by reference in its entirety. [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. TILs are dominated by T cells, 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 research is 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, severely limiting the ability to treat patients who are refractory to other checkpoint inhibitor therapies. 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 a production process for use in generating more robust TILs.

[0004] The present invention provides improved and / or shortened processes and methods for preparing TILs with specific combinations of cytokines and other cell culture media components to prepare therapeutic TIL populations with increased therapeutic efficacy for the treatment of cancer with TILs. Summary of the Invention

[0005] Provided herein are TILs genetically modified to silence or reduce the expression of endogenous PD-1, CD39, and / or CD103. In some embodiments, the subject TILs are produced by genetically engineering a TIL population selected for CD39 and / or CD103 expression (i.e., a TIL population expressing CD39 and / or CD103). Expression of CD39 and CD103 is believed to correlate with PD-1 expression in TILs. TILs that express PD-1 are believed to have enhanced anti-tumor activity. However, PD-1 is known to be immunosuppressive. Also provided herein are expansion methods for producing such genetically modified TILs and therapeutic methods using such TILs.

[0006] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs in a plurality of tumor fragments obtained from a tumor sample excised from the subject's or patient's tumor; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs of (a) to obtain a population of TILs expressing CD39 and / or CD103; and (c) administering a modified population of TILs to the subject or subject. (d) performing a first expansion of the TIL population expressing CD39 and / or CD103 by culturing the TIL population in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system; and (e) performing a second TIL collection. (d) performing a second expansion by culturing the cells in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system, to produce a third TIL population; and (f) the therapeutic TIL population obtained from step (e). (g) transferring the harvested therapeutic TIL population from step (f) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag using a cryopreservation process; (i) administering a therapeutically effective dose of the therapeutic TIL population from the infusion bag of step (h) to the subject; and (j) determining whether the administered therapeutic TIL population is PD-1,and genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (b) selecting the CD39- and / or CD103-positive TILs and prior to (i) administering, so that the TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and / or CD103.

[0007] In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor-infiltrating lymphocytes (TILs), the method comprising: (a) selecting CD39- and / or CD103-positive TILs from a first population of TILs in a tumor digest produced by digesting a tumor sample excised from the tumor in the patient or subject in an enzymatic digestion medium to obtain a population of TILs that express PD-1; and (b) selecting the CD39- and / or CD103-expressing TIL population from a first population of TILs in a tumor digest produced by digesting a tumor sample excised from the tumor in the patient or subject in an enzymatic digestion medium to obtain a population of TILs that express PD-1. (c) performing a first expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain a second TIL population; and (d) performing a second expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days. (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; and (e) transferring the harvested therapeutic TIL population from step (d) to an infusion bag. (f) cryopreserving the infusion bag using a cryopreservation process; (g) administering a therapeutically effective dose of the therapeutic TIL population from the infusion bag of step (f) to the subject; and (h) at any time after (a) selection of CD39 and / or CD103 positive TILs and prior to (g) administration, such that the administered therapeutic TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.and genetically modifying the TIL population that expresses CD39 and / or CD103, the second TIL population, and / or the third TIL population. In some embodiments, step (a) comprises selecting CD39- and / or CD103-positive TILs from a first TIL population in a tumor digest produced by digesting, in an enzymatic digestion medium, multiple tumor fragments prepared from a tumor sample resected from a tumor in a patient or subject, to obtain a TIL population that expresses CD39 and / or CD103.

[0008] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a first population of TILs in a plurality of tumor fragments prepared from a tumor sample excised from the tumor in the patient or subject; (b) selecting CD39- and / or CD103-positive TILs from the first population of TILs of (a) to obtain a population of TILs expressing CD39 and / or CD103; and (c) selecting CD39- and / or CD103-expressing TILs from the first population of TILs. (d) performing a first expansion of the TIL population by culturing the TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system; and (e) producing a second TIL population by culturing the second TIL population in a first cell culture medium supplemented with IL-2, OKT. (d) performing a second expansion by culturing in a second cell culture medium supplemented with -3 and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the system, to produce a third TIL population; and (f) harvesting the third TIL population obtained from step (e), wherein the transition from step (e) to step (f) occurs. (g) transferring the harvested third TIL population from step (f) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag using a cryopreservation process; (i) administering a therapeutically effective dose of the third TIL population from the infusion bag of step (h) to the subject; and (j) administering a therapeutically effective dose of the third TIL population from the infusion bag of step (h) to the subject, such that the administered third TIL population comprises a genetic modification that reduces expression of PD-1, CD39, and / or CD103.and any time after selecting the CD39 and / or CD103 positive TILs and before administering step (i), genetically modifying the TIL population, the second TIL population, and / or the third TIL population to express CD39 and / or CD103.

[0009] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) selecting CD39 and / or CD103 positive TILs from a first population of TILs in a tumor digest produced by digesting a tumor sample excised from the tumor in the patient or subject in an enzymatic digestion medium to obtain a population of TILs expressing CD39 and / or CD103; and (b) selecting the population of TILs expressing CD39 and / or CD103 from a first population of TILs in a tumor digest produced by digesting a tumor sample excised from the tumor in the patient or subject in an enzymatic digestion medium to obtain a population of TILs expressing CD39 and / or CD103. (c) performing a first expansion by culturing the second TIL population in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population; and (c) performing a second expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion (b) is performed for about 7-11 days to obtain a third TIL population, and the second expansion is performed in a sealed container providing a second gas permeable surface area, and the transition from step (b) to step (c) occurs without opening the system, to produce a third TIL population; (d) harvesting the obtained third TIL population from step (c), and the transition from step (c) to step (d) occurs without opening the system; and (e) transferring the harvested third TIL population from step (d) to an infusion bag, and (f) cryopreserving the infusion bag using a cryopreservation process; (g) administering a therapeutically effective dose of a third population of TILs from the infusion bag of step (f) to a subject; and (h) at any time after selecting the CD39 and / or CD103 positive TILs (a) and before administering step (g), such that the administered third population of TILs comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.genetically modifying the TIL population, the second TIL population, and / or the third TIL population to express CD39 and / or CD103;

[0010] In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest produced by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample resected from a patient or subject's tumor to obtain a TIL population expressing CD39 and / or CD103.

[0011] In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs from a cancer in the patient or subject by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells; (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs of (a) to obtain CD39 and / or CD103 positive TILs; (c) obtaining a TIL population expressing CD39 and / or CD103; (d) performing a first expansion of the TIL population expressing CD39 and / or CD103 by culturing the TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed vessel providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (c) to step (d) is performed by converting the system into a closed vessel. (e) performing a second expansion of the second TIL population by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system; and (f) performing a second expansion of the second TIL population by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system. (g) transferring the harvested third TIL population from step (f) to an infusion bag, wherein the transition from step (e) to step (f) occurs without opening the system; (h) cryopreserving the infusion bag using a cryopreservation process; and (i) administering a therapeutically effective dose of the third TIL population from the infusion bag of step (h) to a subject.(j) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after selecting the CD39- and / or CD103-positive TILs and prior to administering step (i), such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0012] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor infiltrating lymphocytes (TILs), the method comprising: (a) removing a tumor sample from a tumor in the subject or patient, the tumor comprising a first population of TILs, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer; (b) processing the tumor sample into a plurality of tumor fragments; and (c) treating the plurality of tumor fragments. (d) enzymatically digesting the TILs in an enzymatic digestion medium to obtain a first TIL population; (d) selecting CD39 and / or CD103-positive TILs from the first TIL population of (c) to obtain a TIL population expressing CD39 and / or CD103; (e) adding the TIL population expressing CD39 and / or CD103 to the closed system; and (f) performing a first expansion by culturing the TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second TIL population. (g) performing a second expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the first expansion is performed in a sealed container providing a first gas-permeable surface area, the first expansion being performed for about 3-11 days to obtain a second TIL population, and the transition from step (e) to step (f) occurring without opening the system; and (g) performing a second expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population. is performed in a sealed container that provides a second gas permeable surface area, and the transition from step (f) to step (g) occurs without opening the system, producing a third TIL population; (h) harvesting the third TIL population obtained from step (g), wherein the transition from step (g) to step (h) occurs without opening the system; and (i) transferring the harvested third TIL population from step (h) to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system.(j) cryopreserving the infusion bag using a cryopreservation process; (k) administering a therapeutically effective dose of the third TIL population from the infusion bag of step (j) to the subject; and (k) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population any time after selecting the CD39- and / or CD103-positive TILs (d) and prior to administering step (i), such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0013] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest produced by digesting in an enzymatic digestion medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in the patient or subject; and (b) selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest produced by digesting in an enzymatic digestion medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in the patient or subject. (b) performing a first expansion of the TIL population expressing CD39 and / or CD103 by culturing the TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population; and (c) performing a first expansion of the second TIL population by culturing the second TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs). (b) culturing the TILs in a second cell culture medium containing the second gas permeable surface area to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, the second expansion being performed in a sealed container that provides a second gas permeable surface area, and the transition from step (b) to step (c) occurs without opening the system, producing a third TIL population; and (d) harvesting the third TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system. (e) transferring the harvested third TIL population from step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; (f) cryopreserving the infusion bag using a cryopreservation process; (g) administering a therapeutically effective dose of the third TIL population from the infusion bag of step (f) to the subject; and (h) administering a therapeutically effective dose of the third TIL population to the subject, such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.and genetically modifying the TIL population, the second TIL population, and / or the third TIL population to express CD39 and / or CD103 any time after selecting the CD39 and / or CD103 positive TILs and prior to administering step (g).

[0014] In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, to produce a TIL population expressing CD39 and / or CD103.

[0015] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from a subject or patient by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells; and (b) isolating CD39- and / or CD103-positive TILs from the first population of TILs of (a). to obtain a TIL population expressing CD39 and / or CD103; and (c) performing an initial expansion (or first expansion by priming) of the TIL population expressing CD39 and / or CD103 in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days to obtain the second TIL population. (d) performing a second rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), and APC, and the rapid expansion is performed for a period of 14 days or less, and optionally, the rapid second expansion can continue 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 a third TIL population; and (e) performing a step of harvesting the third TIL population. (f) administering a therapeutically effective dose of the third TIL population to a subject or patient with cancer; and (g) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (b) selecting the CD39- and / or CD103-positive TILs and before (f) administering, such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0016] In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a tumor sample from the cancer in the subject or patient, the tumor sample comprising a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer; (b) fragmenting the tumor into a plurality of tumor fragments; and (c) fragmenting a first of the plurality of tumor fragments. (d) performing an initial expansion (or first expansion by priming) of the population of TILs expressing CD39 and / or CD103 in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming is performed over a period of 1 to 8 days. (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), 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 initiation of the rapid second expansion; and (f) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population. (g) administering a therapeutically effective dose of the third TIL population to a subject or patient with cancer; and (h) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (c) selecting the CD39- and / or CD103-positive TILs and before (g) administering, such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0017] In another aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) isolating CD39- and / or CD103-positive TILs from a first population of TILs in a tumor digest prepared by digesting in an enzymatic digestion medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from cancer in the patient or subject; (b) selecting TILs to produce a TIL population expressing CD39 and / or CD103; and (b) performing an initial expansion (or first expansion by priming) of the TIL population expressing CD39 and / or CD103 in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days. (c) performing a second rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), 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 initiation of the rapid second expansion to obtain a third TIL population; and (d) harvesting the third TIL population. (e) administering a therapeutically effective dose of the third TIL population to a subject or patient with cancer; and (f) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (a) selection of CD39- and / or CD103-positive TILs and prior to (e) administration, such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, to produce a TIL population expressing CD39 and / or CD103.

[0018] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in the patient or subject; and (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs of (a). (c) performing a first priming expansion by culturing the CD39 and / or CD103-expressing TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas-permeable surface area, and the first priming expansion is performed for a first period of about 3 to 14 days to obtain the second TIL population; (d) restimulating the second TIL population with OKT-3 to produce a second TIL population, wherein the second TIL population is more numerous than the first TIL population; (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces expression of PD-1, CD39, and / or CD103; and (f) culturing the modified second TIL population in a second culture medium supplemented with IL-2, OKT-3, and APC. (g) performing a second rapid expansion by culturing in soil to produce a third TIL population, wherein the second rapid expansion is performed over a second period of time of up to about 14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103; (g) harvesting the therapeutic TIL population; and (h) administering a therapeutically effective portion of the therapeutic TIL population to a subject or patient with cancer.

[0019] In one aspect, provided herein is a method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor-infiltrating lymphocytes (TILs), the method comprising: (a) selecting CD39- and / or CD103-positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in the patient or subject; (b) performing a first priming expansion of the CD39 and / or CD103-expressing TIL population by culturing the CD39 and / or CD103-expressing TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas-permeable surface area, and the first priming expansion is performed for a first period of about 3 to 14 days to obtain the second TIL population. (c) restimulating the second TIL population with OKT-3 to produce a second TIL population, wherein the second TIL population is more numerous than the first TIL population; (d) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification to reduce expression of PD-1, CD39, and / or CD103; and (e) restimulating the second TIL population with OKT-3 supplemented with IL-2, OKT-3, and APCs to produce a modified second TIL population. to produce a third TIL population, wherein the rapid second expansion is performed for a second period of time of not more than about 14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103; (f) harvesting the therapeutic TIL population; and (g) administering a therapeutically effective portion of the therapeutic TIL population to a subject or patient with cancer.In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, to produce a TIL population expressing CD39 and / or CD103.

[0020] In one aspect, provided herein is a method of expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first TIL population in a plurality of tumor fragments prepared from a tumor sample resected from cancer in a subject; (b) selecting CD39- and / or CD103-positive TILs from the first TIL population of step (a) to obtain a TIL population expressing CD39 and / or CD103; and (c) selecting the PD-1-expressing TIL population from a plurality of tumor fragments prepared from a plurality of tumor fragments prepared from a tumor sample resected from cancer in a subject, the plurality of tumor fragments being selected from a plurality of tumor fragments; and (d) selecting the PD-1-expressing TIL population from a plurality of tumor fragments prepared from a plurality of tumor fragments prepared from a tumor sample resected from cancer in a subject, the plurality of tumor fragments being selected ... being selected from a plurality of tumor fragments (d) performing a first primed expansion by culturing the second TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and APC to produce a second TIL population, wherein the first primed expansion is performed in a container comprising a first gas permeable surface area, and the first primed expansion is performed for a first period of about 1-7 / 8 days to obtain a second TIL population, wherein the second TIL population is more numerous than the first TIL population; and (d) culturing the second TIL population in a second culture medium supplemented with IL-2, OKT-3, and APC to produce a second TIL population. (e) performing a second rapid expansion by culturing the therapeutic TILs obtained from step (d) in a container comprising a second gas permeable surface area to produce a third TIL population, wherein the number of APCs added in the second rapid expansion is at least twice the number of APCs added in step (b), and the second rapid expansion is performed over a second period of about 1 to 11 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and the second rapid expansion is performed in a container comprising a second gas permeable surface area. (f) transferring the therapeutic TIL population obtained from step (e) to an infusion bag; and (g) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (b) selection of CD39- and / or CD103-positive TILs and before (f) transfer to the infusion bag, such that the transferred therapeutic TIL population comprises genetically modified TILs comprising genetic modifications that reduce expression of PD-1, CD39, and / or CD103.

[0021] In one aspect, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) selecting CD39- and / or CD103-positive TILs from a first TIL population in a tumor digest obtained from digesting in an enzymatic digestion medium a plurality of tumor fragments prepared from a tumor sample resected from a cancer in a subject, to obtain a TIL population expressing CD39 and / or CD103; and (b) culturing the CD39- and / or CD103-expressing TIL population in a culture medium containing IL-2, OKT-3, and antigen-presenting cells ( (c) performing a first priming expansion by culturing the second TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and APCs to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1-7 / 8 days to obtain a second TIL population, wherein the second TIL population is more numerous than the first TIL population; and (d) performing a second rapid expansion by culturing the therapeutic TILs obtained from step (c) in a medium to produce a third TIL population, wherein the number of APCs added in the second rapid expansion is at least twice the number of APCs added in step (a), and the second rapid expansion is performed over a second period of about 1 to 11 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and the second rapid expansion is performed in a container comprising a second gas permeable surface area; (e) transferring the harvested therapeutic TIL population from step (d) to an infusion bag; and (f) genetically modifying the CD39 and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (a) selection of CD39 and / or CD103-positive TILs and before (e) transfer to the infusion bag, such that the transferred therapeutic TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0022] In another aspect, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining and / or receiving a first TIL population in a plurality of tumor fragments prepared from a tumor sample resected from cancer in a subject or patient; (b) selecting CD39 and / or CD103 positive TILs from the first TIL population of (a) to obtain a TIL population expressing CD39 and / or CD103; and (c) adding the TIL population expressing CD39 and / or CD103 to a closed system. (d) performing a first expansion by culturing the TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system; and (e) producing a second TIL population by culturing the second TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells. (d) performing a second expansion by culturing in a second cell culture medium supplemented with (APC) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the system, producing a third TIL population; and (f) harvesting the therapeutic TIL population obtained from step (e), wherein (g) transferring the harvested therapeutic TIL population from step (f) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; and (h) at any time after selection of CD39 and / or CD103 positive TILs (b) and before transfer to the infusion bag (g), such that the transferred third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.genetically modifying the TIL population, the second TIL population, and / or the third TIL population to express CD39 and / or CD103;

[0023] In another aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium a plurality of tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) performing a first expansion of the TIL population expressing CD39 and / or CD103 by culturing the TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population; and (c) performing a second expansion of the TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs). performing a second expansion by culturing the TIL population to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system to produce a third TIL population; and (d) harvesting the therapeutic TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs. (e) transferring the harvested therapeutic TIL population from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; and (f) at any time after (a) selection of CD39 and / or CD103 positive TILs and before (e) transfer to the infusion bag, such that the transferred third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.genetically modifying the TIL population, the second TIL population, and / or the third TIL population to express CD39 and / or CD103;

[0024] In another aspect, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population in a plurality of tumor fragments prepared from a tumor sample excised from cancer in a subject; (b) selecting CD39- and / or CD103-positive TILs from the first TIL population of (a) to obtain a TIL population expressing CD39 and / or CD103; (c) adding the TIL population expressing CD39 and / or CD103 to a closed system; and (d) administering the TILs to a subject. (c) performing a first expansion by culturing a TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system; and (e) supplementing the second TIL population with IL-2, OKT-3, and antigen presenting cells (APCs). (d) culturing the TILs in a second cell culture medium containing the second gas permeable surface area to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, the second expansion being performed in a sealed container providing a second gas permeable surface area, and the transition from step (d) to step (e) occurring without opening the system; and (f) harvesting the third TIL population obtained from step (e), wherein the transition from step (e) to step (f) occurring without opening the system. (g) transferring the collected third TIL population from step (f) into an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; and (h) selecting the CD39 and / or CD103 positive TIL population at any time after (b) and before (g) transferring the CD39 and / or CD103 expressing TIL population into the infusion bag, such that the transferred third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.and genetically modifying the second and / or third TIL populations.

[0025] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest produced by digesting a tumor sample excised from a cancer in a patient or subject in an enzymatic digestion medium to obtain a TIL population expressing CD39 and / or CD103; and (b) selecting the CD39 and / or CD103 expressing TIL population from a first TIL population supplemented with IL-2. (c) performing a first expansion by culturing the second TIL population in a culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas-permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population; and (c) performing a second expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 3 to 11 days to obtain the second TIL population. (d) harvesting the harvested third TIL population from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; and (e) transferring the harvested third TIL population from step (d) to an infusion bag. wherein the transition from step (d) to (e) occurs without opening the system; and (f) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (a) selection of CD39- and / or CD103-positive TILs and before (e) transfer to the infusion bag, such that the transferred third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest produced by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample resected from cancer in a patient or subject, to obtain a TIL population expressing CD39 and / or CD103.

[0026] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; and (b) selecting CD39 and / or CD103 positive TILs from the first population of TILs of (a) to obtain a TIL population that expresses CD39 and / or CD103. (c) adding a population of TILs expressing CD39 and / or CD103 to the closed system; and (d) performing a first expansion by culturing the population of TILs expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2 to produce a second population of TILs, 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 11 days to obtain the second population of TILs, and the transition from step (c) to step (d) occurs without opening the system. (e) performing a second expansion of the second TIL population by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain a third TIL population, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system; and (f) performing a second expansion of the third TIL population obtained from step (e). (g) transferring the harvested third TIL population from step (f) into an infusion bag, wherein the transition from step (e) to step (f) occurs without opening the system; and (h) after selection of CD39 and / or CD103 positive TILs (b), such that the transferred third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39 and / or CD103.and (g) genetically modifying the TIL population, the second TIL population, and / or the third TIL population to express CD39 and / or CD103 at any time prior to transfer to the infusion bag.

[0027] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) removing a tumor sample from a cancer in a subject or patient, the tumor sample comprising a first TIL population, optionally by surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer; (b) processing the tumor sample into a plurality of tumor fragments; and (c) enzymatically digesting the plurality of tumor fragments in an enzymatic digestion medium to extract the first TIL population. (d) selecting CD39 and / or CD103 positive TILs from the first TIL population of (c) to obtain a TIL population expressing CD39 and / or CD103; (e) adding the TIL population expressing CD39 and / or CD103 to the closed system; and (f) performing a first expansion of the TIL population expressing CD39 and / or CD103 by culturing it in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a dense cell culture medium that provides a first gas permeable surface area. (g) performing a second expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and the first expansion is performed for about 3-11 days to obtain a second TIL population, and the transition from step (e) to step (f) occurs without opening the system; and (g) performing a second expansion by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, and the second expansion is performed in a closed container providing a second gas-permeable surface area. (h) harvesting the third TIL population obtained from step (g), wherein the transition from step (g) to step (h) occurs without opening the system; (i) transferring the harvested third TIL population from step (h) to an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; and (j) detecting that the transferred third TIL population is PD-1,and genetically modifying the CD39 and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (d) selection of CD39 and / or CD103-positive TILs and before (h) transfer to the infusion bag, so that the TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of CD39 and / or CD103.

[0028] In another aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) performing a first expansion of a TIL population expressing CD39 and / or CD103 by culturing the TIL population in a first cell culture medium supplemented with IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 11 days to obtain the second TIL population; (c) performing a second expansion of the second TIL population by culturing the second TIL population in a second cell culture medium supplemented with IL-2, OKT-3, and antigen presenting cells (APCs), and a third expansion of the second TIL population. (d) harvesting the third TIL population obtained from step (c), wherein the second expansion is performed for about 7-11 days to obtain a third TIL population, the second expansion being performed in a sealed container that provides a second gas permeable surface area, and the transition from step (b) to step (c) occurring without opening the system; (d) harvesting the third TIL population obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system; and (e) harvesting the harvested third TIL population from step (d). into an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; and (f) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (a) selection of CD39- and / or CD103-positive TILs and prior to (e) transfer to the infusion bag, such that the transferred third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, to produce a TIL population expressing CD39 and / or CD103.

[0029] In another aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising the steps of: (a) obtaining and / or receiving a first population of TILs in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a subject or patient; (b) selecting CD39 and / or CD103 positive TILs from the first TIL population of (a) to identify CD39 and / or CD103 positive TILs; (c) performing an initial expansion (or first expansion by priming) of the TIL population expressing CD39 and / or CD103 in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days. (d) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (anti-CD3 antibody), 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 initiation of the rapid second expansion. (b) obtaining a population of CD39- and / or CD103-positive TILs; (c) obtaining a population of CD39- and / or CD103-expressing TILs; (d) obtaining a population of CD39- and / or CD103-expressing TILs; (e) harvesting a third TIL population; and (f) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (b) selection of CD39- and / or CD103-positive TILs and before (f) harvesting such that the harvested third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0030] In one aspect, provided herein is a method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a tumor sample from a cancer in a subject or patient, the tumor sample including a first TIL population, optionally via surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer; (b) fragmenting the tumor sample into a plurality of tumor fragments; and (c) isolating C16 cells from the first TIL population in the tumor fragments. (d) selecting CD39- and / or CD103-positive TILs to obtain a population of TILs expressing CD39 and / or CD103; and (d) performing an initial expansion (or first expansion by priming) of the population of TILs expressing CD39 and / or CD103 in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming is performed in a manner similar to that described above. (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (an anti-CD3 antibody), and APCs, 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. (c) obtaining a third TIL population capable of expressing CD39 and / or CD103; (f) harvesting the third TIL population; and (g) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (c) selecting CD39- and / or CD103-positive TILs and before (f) harvesting, such that the harvested third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0031] In another aspect, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) producing a population of TILs expressing CD39 and / or CD103; and (b) performing an initial expansion (or first expansion by priming) of the population of TILs expressing CD39 and / or CD103 in a first cell culture medium to obtain a second population of TILs, wherein the first cell culture medium is supplemented with IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days. (c) performing a second rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium is supplemented with IL-2, OKT-3 (an anti-CD3 antibody), and APCs, 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 initiation of the rapid second expansion. (d) harvesting a third TIL population; and (e) genetically modifying the CD39- and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any time after (a) selection of CD39- and / or CD103-positive TILs and before (d) harvesting, such that the harvested third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0032] In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, to produce a TIL population expressing CD39 and / or CD103.

[0033] In one aspect, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first TIL population in a plurality of tumor fragments prepared from a tumor sample resected from a subject's cancer; (b) enzymatically digesting the plurality of tumor fragments in an enzyme digestion medium to obtain a first TIL population; (c) selecting PD-1-positive TILs from the first TIL population of step (b) to obtain a TIL population expressing CD39 and / or CD103; and (d) performing a first expansion by priming by culturing the CD39- and / or CD103-expressing TIL population in a first cell culture medium supplemented with IL-2, an anti-CD3 agonist antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion by priming is performed over a first period of about 1 to 11 days to obtain a second TIL population, and (e) restimulating the second TIL population with an anti-CD3 agonist antibody; (f) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces expression of PD-1, CD39, and / or CD103; and (g) generating the modified second TIL population. (h) performing a second rapid expansion of the population by culturing the population in a second cell culture medium supplemented with IL-2, an anti-CD3 agonist antibody, and APCs to produce a third TIL population, wherein the second rapid expansion is performed over a second period of about 1 to 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; and (h) harvesting the therapeutic TIL population obtained from step (g).

[0034] In certain embodiments, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) selecting CD39- and / or CD103-positive TILs from a first TIL population in a tumor digest prepared by enzymatically digesting, in an enzymatic digestion medium, multiple tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, to produce a TIL population expressing CD39 and / or CD103; and (b) performing a first expansion by priming by culturing the CD39- and / or CD103-expressing TIL population in a first cell culture medium supplemented with IL-2, an anti-CD3 agonist antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first expansion by priming is performed over a first period of about 1 to 11 days. (c) restimulating the second TIL population with an anti-CD3 agonist antibody to produce a second TIL population, the second TIL population being more numerous than the first TIL population; (d) genetically modifying the second TIL population to produce a modified second TIL population, the modified second TIL population comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103; and (e) (f) performing a second rapid expansion of the modified second TIL population by culturing it in a second culture medium supplemented with IL-2, an anti-CD3 agonist antibody, and APCs to produce a third TIL population, wherein the second rapid expansion is performed over a second period of about 1 to 11 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; and (f) harvesting the therapeutic TIL population obtained from step (e). In some embodiments, in step (d), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (e) is greater than the number of APCs in the culture medium in step (d).

[0035] In another aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first TIL population in a tumor sample obtained from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) enzymatically digesting the tumor sample in an enzymatic digestion medium to obtain the first TIL population; and (c) extracting TILs from the first TIL population of (b). (d) performing a first priming expansion of the PD-1-expressing TIL population by culturing the PD-1-expressing TIL population in a first cell culture medium supplemented with IL-2, an anti-CD3 agonist antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas-permeable surface area, and the first priming expansion is performed for about 3 days. (e) restimulating the second TIL population with an anti-CD3 agonist antibody; and (f) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces expression of PD-1, CD39, and / or CD103. (g) performing a second rapid expansion of the modified second TIL population by culturing it in a second culture medium supplemented with IL-2, an anti-CD3 agonist antibody, and APCs to produce a third TIL population, wherein the second rapid expansion is performed over a second period of time of up to about 14 days to obtain a third TIL population, wherein the third TIL population comprises a genetic modification that reduces expression of PD-1, CD39, and / or CD103, producing a third TIL population; and (h) harvesting the third TIL population.

[0036] In one aspect, provided herein is a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) enzymatically digesting a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, in an enzymatic digestion medium, to prepare a first TIL population in a tumor digest; and (b) selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest to obtain a first TIL population. (b) performing a first priming expansion of the PD-1-expressing TIL population by culturing the PD-1-expressing TIL population in a first cell culture medium supplemented with IL-2, an anti-CD3 agonist antibody, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed over a first period of about 3 to 14 days. (c) obtaining two populations of TILs, the second population of TILs being more numerous than the first population of TILs; (d) restimulating the second population of TILs with an anti-CD3 agonist antibody; (e) genetically modifying the second population of TILs to produce a modified second population of TILs, the modified second population of TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103; and (f) generating the modified second population of TILs. (f) performing a second rapid expansion of the L population by culturing the L population in a second culture medium supplemented with IL-2, an anti-CD3 agonist antibody, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed over a second period of time of up to about 14 days to obtain a third TIL population, wherein the third TIL population comprises a genetic modification that reduces expression of PD-1, CD39, and / or CD103; and (f) harvesting the third TIL population.In some embodiments, step (a) comprises selecting CD39 and / or CD103 positive TILs from a first TIL population in a tumor digest prepared by digesting in an enzymatic digestion medium multiple tumor fragments prepared from a tumor sample obtained or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject, to produce a TIL population expressing CD39 and / or CD103.

[0037] In some embodiments, the anti-CD3 agonist antibody is OKT-3.

[0038] In some embodiments of the subject methods, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

[0039] In one aspect, provided herein is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first priming expansion by culturing a first TIL population expressing CD39 and / or CD103 in a first cell culture medium supplemented with IL-2, optionally OKT-3, and optionally comprising antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed over a first period of about 1 to 11 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population; and (b) performing a first priming expansion by culturing the second TIL population in a first cell culture medium supplemented with IL-2, OKT-3, and APCs to produce a second TIL population. and (c) genetically modifying the TIL population, the second TIL population, and / or the third TIL population any time prior to harvesting step (c) such that the harvested third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers. In some embodiments, in step (a), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

[0040] In another aspect, provided herein is a method of expanding T cells, the method comprising: (a) performing a first expansion by priming a first TIL population obtained from a donor by culturing the first TIL population to result in growth and priming activation of a first T cell population, wherein the first TIL population is a TIL population that expresses at least CD39 and CD103; and (b) after activation of the first T cell population primed in step (a) begins to decline, performing a first expansion by culturing the first TIL population to result in growth and priming activation of a first T cell population. (c) performing a rapid second expansion of the first TIL population by culturing the first TIL population to result in growth and promote activation of the first T cell population to obtain a second T cell population; (d) genetically modifying the first TIL population and / or the second TIL population such that the harvested second TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers.

[0041] In one aspect, provided herein is a method of expanding T cells, comprising: (a) performing a first expansion by priming a first T cell population from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor in a donor by culturing the first T cell population to result in growth and priming activation of the first T cell population, wherein the first T cell population is a T cell population that expresses at least CD39 and CD103; and (b) measuring the activity of the first T cell population primed in step (a). (c) performing a rapid second expansion of the first TIL population by culturing the first TIL population after T cell exhaustion begins to decline, resulting in growth and promoting activation of the first T cell population to obtain a second T cell population; (d) genetically modifying the first TIL population and / or the second TIL population such that the harvested second T cell population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers.

[0042] In some embodiments, the one or more T cell exhaustion markers are selected from PD-1 and CD39, and combinations thereof, hi some embodiments, the one or more factors phenotypically associated with the T cell exhaustion marker is CD103.

[0043] In another aspect, the methods described herein include an additional selection step. In some embodiments, the method further includes second selecting, from the TIL population, a TIL population that expresses at least one protein selected from the group consisting of OX40, 4-1BB, and combinations thereof. In some embodiments, the second selection occurs before the initiation of the first expansion. In some embodiments, the second selection occurs within 5 days of the initiation of the first expansion, and OKT-3 has not yet been added. In some embodiments, the method further includes second selecting, from the second TIL population, a TIL population that expresses at least one protein selected from the group consisting of OX40, 4-1BB, and combinations thereof.

[0044] In some embodiments, the modification is performed on the second TIL population from the first expansion, or the third TIL population from the second expansion, or both. In some embodiments, the modification is performed on the second TIL population from the first expansion by priming, or the third TIL population from the rapid second expansion, or both. In some embodiments, the modification is performed on the second TIL population from the first expansion and the second TIL population before the second expansion. In some embodiments, the modification is performed on the second TIL population from the first expansion by priming and the second TIL population before the rapid second expansion. In some embodiments, the modification is performed on the third TIL population from the second expansion. In some embodiments, the modification is performed on the third TIL population from the rapid second expansion. In some embodiments, the modification is performed after harvesting.

[0045] In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the first expansion with priming is performed over a period of about 11 days.

[0046] In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first expansion by priming.

[0047] In some embodiments, in the second expansion step, IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL. In some embodiments, in the rapid second expansion step, IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0048] In some embodiments, the first expansion is performed using a gas-permeable container. In some embodiments, the first expansion by priming is performed using a gas-permeable container. In some embodiments, the second expansion is performed using a gas-permeable container. In some embodiments, the rapid second expansion is performed using a gas-permeable container.

[0049] In some embodiments, the cell culture medium of the first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0050] In some embodiments, the cell culture medium of the first expansion by priming further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0051] In some embodiments, the cell culture medium of the second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0052] In some embodiments, the rapid secondary expansion cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0053] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the therapeutic TIL population to the patient.

[0054] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m 2 Fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 1 day.

[0055] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 5 days.

[0056] In some embodiments, the method further comprises administering cyclophosphamide with mesna.

[0057] In some embodiments, the method further comprises treating the patient with an IL-2 regimen beginning the day after administration of the TILs to the patient.

[0058] In some embodiments, the method further comprises treating the patient with an IL-2 regimen starting on the same day that the TILs are administered to the patient.

[0059] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0060] In some embodiments, the therapeutically effective TIL population is about 2.3 x 10 10 ~Approx. 13.7×10 10 TIL.

[0061] In some embodiments, the priming first expansion and the rapid second expansion are performed over a period of 21 days or less. In certain embodiments, the priming first expansion and the rapid second expansion are performed over a period of 16 or 17 days or less. In certain embodiments, the priming first expansion is performed over a period of 7 or 8 days or less. In certain embodiments, the rapid second expansion is performed over a period of 11 days or less. In some embodiments, the priming first expansion and the rapid second expansion are each performed independently within a period of 11 days.

[0062] In some embodiments of this method, all steps are performed within about 26 days. In certain embodiments, the first cell culture medium and the second cell culture medium are different. In some embodiments, the first cell culture medium and the second cell culture medium are the same.

[0063] In some embodiments, about 4 or 5 days after the initiation of the rapid second expansion, the culture is split into multiple passage cultures and cultured for about 6 or 7 days in a third culture medium supplemented with IL-2 to produce a third TIL population.

[0064] In certain embodiments, a first expansion by priming is performed in a sealed container comprising a first gas permeable surface area, a second rapid expansion is initiated in a sealed container comprising a second gas permeable surface area, and multiple subcultures are cultured in multiple sealed containers comprising a third gas permeable surface area.

[0065] In some embodiments, the second TIL population is transferred from the sealed container comprising the first gas permeable surface area to the sealed container comprising the second gas permeable surface area without opening the system, the second TIL population is transferred from the sealed container comprising the second gas permeable surface area to a plurality of sealed containers comprising a third gas permeable surface area without opening the system, and the third TIL population is harvested from the plurality of sealed containers comprising the third gas permeable surface area without opening the system.

[0066] In some embodiments, about 4 or 5 days after the initiation of the second expansion, the culture is split into multiple sealed subculture vessels, each containing a third gas permeable surface area, and cultured in a third cell culture medium supplemented with IL-2 for about 6 or 7 days to produce a third TIL population.

[0067] In certain embodiments, dividing the culture into multiple sealed subculture vessels results in the transfer of the culture from the sealed vessel comprising the second gas permeable surface to the multiple subculture vessels without opening the system.

[0068] In certain embodiments, the genetically modified TILs express any of the following: CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMA The method further comprises an additional genetic modification to reduce expression of one or more immune checkpoint genes selected from the group including: D2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. In an exemplary embodiment, the one or more immune checkpoint genes are selected from the group including: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

[0069] In some embodiments, the genetically modified TILs further comprise an additional genetic modification that enhances expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population, wherein the immune checkpoint gene(s) are selected from the group including CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or NOTCH ligand mDLL1.

[0070] In certain embodiments, the genetic modification step is performed on the second TIL population prior to the initiation of the second expansion or rapid second expansion, and the method includes restimulating the second TIL population with OKT-3 for about 2 days prior to performing the genetic modification step.

[0071] In some embodiments, the modified second TIL population is allowed to rest for about 1 day after the genetic modification step and before the initiation of the second expansion or rapid second expansion.

[0072] In some embodiments, the genetic modification step is carried out using a programmable nuclease that mediates the generation of a double- or single-strand break in the one or more immune checkpoint genes.

[0073] In some embodiments, the genetic modification step is performed using one or more methods selected from CRISPR technology, TALE technology, zinc finger technology, and combinations thereof. In some embodiments, the genetic modification step is performed using CRISPR technology. In some embodiments, the CRISPR technology is CRISPR / Cas9 technology. In some embodiments, the genetic modification step is performed using TALE technology. In some embodiments, the genetic modification step is performed using zinc finger technology.

[0074] In some embodiments, processing a tumor sample obtained from a subject into a tumor digest comprises incubating the tumor sample in an enzyme medium. In some embodiments, processing a tumor sample obtained from a subject into a tumor digest further comprises mechanically disrupting the tumor sample to dissociate the tumor sample. In some embodiments, processing a tumor sample obtained from a subject into a tumor digest further comprises purifying the dissociated tumor sample using density gradient separation.

[0075] In some embodiments, the tumor sample or multiple tumor fragments are digested in an enzyme digestion medium prior to the selection step to produce a tumor digest comprising a first TIL population. In some embodiments, the enzyme digestion medium comprises a mixture of enzymes. In some embodiments, the enzyme digestion medium comprises collagenase, neutral protease, and DNase. In some embodiments, the enzyme digestion medium comprises 30 units / mL of DNase. In some embodiments, the enzyme digestion medium comprises collagenase. In some embodiments, the enzyme digestion medium comprises 1.0 mg / mL of collagenase. In some embodiments, the enzyme digestion medium comprises DNase. In some embodiments, the enzyme digestion medium comprises neutral protease. In some embodiments, the enzyme digestion medium comprises hyaluronidase.

[0076] In some embodiments, the tumor sample or multiple tumor fragments are subjected to mechanical dissociation before, during, and / or after digestion of the tumor sample or multiple tumor fragments.

[0077] In one aspect, the methods and compositions disclosed herein describe therapeutic TIL populations. In some embodiments, the harvested therapeutic TIL population comprises sufficient TILs for use in administering a therapeutically effective dose to a subject. In some embodiments, the therapeutically effective dose is about 1 x 10 9 ~Approx. 9×10 10 In some embodiments, the harvested therapeutic TIL population exhibits an increased subpopulation of CD8+ cells compared to the first and / or second TIL populations.

[0078] In some embodiments, the APCs comprise peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are supplemented at a TIL:PBMC ratio of about 1:25.

[0079] In some embodiments, the first expansion and the second expansion are each performed separately within a period of 11 to 12 days. In some embodiments, steps (a)-(e), (f), or (g) are performed in about 10 to about 24 days. In some embodiments, steps (a)-(e), (f), or (g) are performed in about 15 to about 24 days. In some embodiments, steps (a)-(e), (f), or (g) are performed in about 20 to about 24 days. In some embodiments, steps (a)-(e), (f), or (g) are performed in about 20 to about 22 days.

[0080] In some embodiments, the second TIL population is at least 50-fold more numerous than the first TIL population.

[0081] In one aspect, the disclosure includes a TIL population according to any of the above methods. [Brief explanation of the drawings]

[0082] [Figure 1] Illustrative Process 2A chart providing an overview of steps A-F. [Figure 2A] Process flow chart for Process 2A. [Figure 2B] Process flow chart for Process 2A. [Figure 2C] Process flow chart for Process 2A. [Figure 3] FIG. 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] 1 shows a diagram of an embodiment of Process 2A, a 22-day TIL manufacturing process. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Process 2A. [Figure 6] Detailed comparison of Process 1C embodiment and Process 2A embodiment. [Figure 7] Exemplary GEN3-type processes in tumors. [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 Gen3 process chart providing an overview of steps A-F (approximately a 14- to 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] A comparison of embodiments of the 2A process (approximately a 22 day process) and the Gen3 process (approximately a 14-22 day process) for TIL fabrication is shown. [Figure 8F] Chart of an exemplary process CD39-103 Gen3 providing an overview of steps A-F (approximately 14-22 day process). [Figure 8G] A chart of exemplary process OX4-1 Gen3 providing an overview of steps A-F (approximately 14-22 day process). [Figure 9] An experimental flow chart is provided for the comparability between GEN2 (Process 2A) and GEN3. [Figure 10] 1 shows a comparison of various Gen2 (2A process) 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, with the cylinders representing individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains, e.g., derived from antibodies that bind 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 capable of bringing 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 (Gen3.1 Test) of the Gen3.1 Process (16-day Process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 Test (Optimized Gen3.1) process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23A] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process, with exemplary differences highlighted. [Figure 23B]1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process, with exemplary differences highlighted. [Figure 24] FIG. 1 is a schematic diagram of an exemplary embodiment of a 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 (Gen3-optimized, 16-17 day process) are shown. [Figure 33] FIG. 1 is a schematic diagram of an exemplary workflow in the TIL generation process, generally emphasizing selection (e.g., of cells expressing CD39 and / or CD103) prior to expansion. [Figure 34A] Summary of two independent experiments in which 22 tumor samples were analyzed for markers associated with activation and exhaustion (Experiment 1: 12 tumor digests from 5 melanoma, 3 HNSCC, 1 cervix, 1 ovary, 1 prostate, and 1 NSCLC; Experiment 2: 10 tumor digests from 1 melanoma, 3 HNSCC, 1 cervix, 2 ovary, and 3 NSCLC). [Figure 34B]An illustration of a biased approach to identify a TIL population for selection based on unsupervised clustering by PD-1. In this example, PD-1 selected TILs are the baseline for selection with other factors that cluster with them. [Figure 35A] Evaluation of 21 tumor digests (3 cervical, 6 HNSCC, 3 NSCLC, 5 melanoma, 3 ovarian, and 1 prostate) for phenotypic expression of markers associated with activation, exhaustion, and tumor specificity. Tumor digests were gated on PD-1 high, PD-1 intermediate, and PD-1 negative TILs and compared with the CD3+ population. [Figure 35B] Evaluation of 21 tumor digests (3 cervical, 6 HNSCC, 3 NSCLC, 5 melanoma, 3 ovarian, and 1 prostate) for phenotypic expression of markers associated with activation, exhaustion, and tumor specificity. Tumor digests were gated on PD-1 high, PD-1 intermediate, and PD-1 negative TILs and compared with the CD3+ population. [Figure 35C] Evaluation of 21 tumor digests (3 cervical, 6 HNSCC, 3 NSCLC, 5 melanoma, 3 ovarian, and 1 prostate) for phenotypic expression of markers associated with activation, exhaustion, and tumor specificity. Tumor digests were gated on PD-1 high, PD-1 intermediate, and PD-1 negative TILs and compared with the CD3+ population. [Figure 35D] Evaluation of 21 tumor digests (3 cervical, 6 HNSCC, 3 NSCLC, 5 melanoma, 3 ovarian, and 1 prostate) for phenotypic expression of markers associated with activation, exhaustion, and tumor specificity. Tumor digests were gated on PD-1 high, PD-1 intermediate, and PD-1 negative TILs and compared with the CD3+ population. [Figure 35E] Evaluation of 21 tumor digests (3 cervical, 6 HNSCC, 3 NSCLC, 5 melanoma, 3 ovarian, and 1 prostate) for phenotypic expression of markers associated with activation, exhaustion, and tumor specificity. Tumor digests were gated on PD-1 high, PD-1 intermediate, and PD-1 negative TILs and compared with the CD3+ population. [Figure 36A]Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36B] Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36C] Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36D] Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36E] Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36F] Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36G] Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36H]Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 36I] Exemplary cluster analysis of 12 tumor digests (5 melanoma, 3 HNSCC, 1 cervical, 1 ovarian, 1 prostate, 1 NSCLC) showing the phenotypic profiles of various tumors (A–D) and the clustering of specific markers (E–I). [Figure 37] Summary of various phenotypic markers in either Ki67- or Ki67+ CD4 and CD8 TILs. [Figure 38A] In vitro expansion of PD-1 high CD39+ selected TILs. [Figure 38B] In vitro expansion of PD-1 high CD39+ selected TILs. [Figure 38C] In vitro expansion of PD-1 high CD39+ selected TILs. [Figure 38D] In vitro expansion of PD-1 high CD39+ selected TILs. [Figure 39] Expression of CD3 and phenotypic markers defining T cell lineage, memory, and differentiation by PD-1 high CD39+ selected TILs. [Figure 40] A-C, Expression of CD3 and phenotypic markers defining T cell activation and exhaustion by PD-1 high CD39+ selected TILs. [Figure 41] IFNγ secretion by PD-1 high CD39+ selected TILs. (Note: H3099, H3117, and CC10052 are not included.) [Figure 42] Tumor reactivity of PD-1-high CD39+ selected TILs.

[0083] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0084] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0085] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0086] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0087] SEQ ID NO: 5 is an IL-2 form.

[0088] SEQ ID NO: 6 is an IL-2 form.

[0089] SEQ ID NO: 7 is an IL-2 form.

[0090] SEQ ID NO: 8 is a mucin domain polypeptide.

[0091] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0092] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0093] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0094] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0095] SEQ ID NO: 13 is the IL-2 sequence.

[0096] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0097] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0098] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0099] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0100] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0101] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0102] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0103] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0104] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0105] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0106] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0107] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0108] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0109] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0110] SEQ ID NO: 28 is the VH chain of IgG.IL2R67A.H1.

[0111] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0112] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0113] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0114] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0115] SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

[0116] SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

[0117] SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

[0118] SEQ ID NO: 36 is the VL chain.

[0119] SEQ ID NO: 37 is the light chain.

[0120] SEQ ID NO: 38 is the light chain.

[0121] SEQ ID NO: 39 is the light chain.

[0122] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0123] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0124] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0125] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0126] SEQ ID NO: 44 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0127] SEQ ID NO: 45 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0128] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0129] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0130] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0131] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0132] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0133] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0134] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0135] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0136] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0137] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0138] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0139] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0140] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0141] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0142] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0143] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0144] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0145] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0146] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0147] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0148] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0149] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0150] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0151] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0152] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0153] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0154] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0155] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0156] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0157] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0158] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0159] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0160] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0161] SEQ ID NO: 79 is the heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0162] SEQ ID NO: 80 is the light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0163] SEQ ID NO: 81 is the heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 2.

[0164] SEQ ID NO: 82 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0165] SEQ ID NO: 83 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E3-2.

[0166] SEQ ID NO: 84 is the light chain variable region (VL) of the 4-1BB agonist antibody H39E3-2.

[0167] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0168] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0169] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0170] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0171] SEQ ID NO: 89 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0172] SEQ ID NO: 90 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0173] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0174] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0175] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0176] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0177] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0178] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0179] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0180] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0181] SEQ ID NO: 99 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 11D4.

[0182] SEQ ID NO: 100 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 11D4.

[0183] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0184] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0185] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0186] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0187] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0188] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0189] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0190] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0191] SEQ ID NO: 109 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 18D8.

[0192] SEQ ID NO: 110 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 18D8.

[0193] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0194] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0195] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0196] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0197] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0198] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0199] SEQ ID NO: 117 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu119-122.

[0200] SEQ ID NO: 118 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu119-122.

[0201] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0202] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0203] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0204] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0205] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0206] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0207] SEQ ID NO: 125 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu106-222.

[0208] SEQ ID NO: 126 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu106-222.

[0209] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0210] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0211] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0212] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0213] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0214] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0215] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0216] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0217] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0218] SEQ ID NO: 136 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 008.

[0219] SEQ ID NO: 137 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 008.

[0220] SEQ ID NO: 138 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 011.

[0221] SEQ ID NO: 139 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 011.

[0222] SEQ ID NO: 140 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 021.

[0223] SEQ ID NO: 141 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 021.

[0224] SEQ ID NO: 142 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 023.

[0225] SEQ ID NO: 143 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 023.

[0226] SEQ ID NO: 144 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0227] SEQ ID NO: 145 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0228] SEQ ID NO: 146 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0229] SEQ ID NO: 147 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0230] SEQ ID NO: 148 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0231] SEQ ID NO: 149 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0232] SEQ ID NO: 150 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0233] SEQ ID NO: 151 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0234] SEQ ID NO: 152 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0235] SEQ ID NO: 153 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0236] SEQ ID NO: 154 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0237] SEQ ID NO: 155 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0238] SEQ ID NO: 156 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0239] SEQ ID NO: 157 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0240] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0241] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0242] SEQ ID NO: 160 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor nivolumab.

[0243] SEQ ID NO: 161 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor nivolumab.

[0244] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0245] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0246] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0247] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0248] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0249] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0250] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0251] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0252] SEQ ID NO: 170 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0253] SEQ ID NO: 171 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0254] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0255] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0256] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0257] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0258] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0259] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0260] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0261] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0262] SEQ ID NO: 180 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor durvalumab.

[0263] SEQ ID NO: 181 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor durvalumab.

[0264] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0265] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0266] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0267] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0268] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0269] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0270] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0271] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0272] SEQ ID NO: 190 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor avelumab.

[0273] SEQ ID NO: 191 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor avelumab.

[0274] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0275] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0276] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0277] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0278] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0279] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0280] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0281] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0282] SEQ ID NO: 200 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0283] SEQ ID NO: 201 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0284] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0285] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0286] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0287] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0288] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0289] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0290] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0291] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0292] SEQ ID NO: 210 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0293] SEQ ID NO: 211 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0294] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0295] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0296] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0297] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0298] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0299] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0300] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0301] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0302] SEQ ID NO: 220 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0303] SEQ ID NO: 221 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0304] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0305] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0306] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0307] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0308] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0309] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0310] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0311] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0312] SEQ ID NO: 230 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0313] SEQ ID NO: 231 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0314] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0315] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0316] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0317] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0318] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0319] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0320] SEQ ID NO: 238 is the target PD-1 sequence.

[0321] SEQ ID NO: 239 is the target PD-1 sequence.

[0322] SEQ ID NO: 240 is a repeated PD-1 left repeat sequence.

[0323] SEQ ID NO: 241 is a repeated PD-1 right repeat sequence.

[0324] SEQ ID NO: 242 is a repeated PD-1 left repeat sequence.

[0325] SEQ ID NO: 243 is a repeated PD-1 right repeat sequence.

[0326] SEQ ID NO: 244 is the PD-1 left TALEN nuclease sequence.

[0327] SEQ ID NO: 245 is the PD-1 right TALEN nuclease sequence.

[0328] SEQ ID NO: 246 is the PD-1 left TALEN nuclease sequence.

[0329] SEQ ID NO: 247 is the PD-1 right TALEN nuclease sequence.

[0330] 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. DETAILED DESCRIPTION OF THE INVENTION

[0331] I. Introduction Provided herein are TILs that express CD39, CD103, and / or both. In some embodiments, the subject TILs are produced by genetically engineering a population of TILs selected for expression of (i) PD-1, (ii) CD39, (iii) CD103, (iv) a combination of (i) and (ii), (v) a combination of (i) and (iii), or (vi) a combination of (ii) and (iii). Expansion methods for producing such TIL populations and therapeutic methods using such TILs are also provided herein.

[0332] 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.

[0333] As used herein, the terms "co-administration," "co-administering," "administered in combination with," "administering in combination with," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., multiple TILs) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Concurrent 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.

[0334] The term "in vivo" refers to events that take place inside a subject's body.

[0335] 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.

[0336] 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.

[0337] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably by at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0338] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream 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.

[0339] 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 the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by efficacy; 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] [Table 1]

[0354] 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.

[0355] 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-xylene sulfonic 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), sulfos ... Fosuccinimidyl-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-(ρ-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.

[0356] 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.

[0357] In some embodiments, a form of IL-2 suitable for use in the present invention is nembulkin alfa, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Inc. Nembulkin alfa is a nucleotide sequence containing 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 );G 2 Human interleukin-2 (IL-2) (4-74)-peptide (62-132) was fused via a peptide linker (60-61) to human interleukin-2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) via a GSG3S peptide linker (133-138). The peptide was produced in Chinese hamster ovary (CHO) cells and was alpha-glycosylated. 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. 2021 / 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.

[0358] [Table 2-1] [Table 2-2]

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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 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 substitutes 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.

[0365] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0366] 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).

[0367] 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).

[0368] 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).

[0369] 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).

[0370] 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 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. Tumor 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.

[0371] 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.

[0372] 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.

[0373] 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.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] 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).

[0379] 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.

[0380] 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.

[0381] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left a subject's bloodstream 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).

[0382] 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 the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by efficacy; 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.

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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."

[0388] 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, termed 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.

[0389] 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 by 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.

[0390] 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.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.

[0396] 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."

[0397] 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.

[0398] The terms "humanized antibody," "humanized antibodies," and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein can also be modified to use any Fc variant known to confer improved (e.g., reduced) effector function and / or FcR binding.Fc variants are described in, for example, International Patent Application Publication Nos. WO1988 / 07089A1, WO1996 / 14339A1, WO1998 / 05787A1, WO1998 / 23289A1, WO1999 / 51642A1, WO99 / 58572A1, WO2000 / 09560A2, WO2000 / 32767A1, WO2000 / 42072A2, and WO2002 / 4 4215A2, WO2002 / 060919A2, WO2003 / 074569A2, WO2004 / 016750A2, WO2004 / 029207A2, WO2004 / 03 5752A2, WO2004 / 063351A2, WO2004 / 074455A2, WO2004 / 099249A2, WO2005 / 040217A2, WO2005 / 07 0963A1, WO2005 / 077981A2, WO2005 / 092925A2, WO2005 / 123780A2, WO2006 / 019447A1, WO2006 / 047350A2, and WO2006 / 085967A2, as well as U.S. Pat. Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096 ,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784 (the disclosures of which are incorporated herein by reference).

[0399] 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.

[0400] A "diabody" is a small antibody fragment that has two antigen-binding sites. The fragments bind to the same polypeptide chain (VH -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). When a linker that is too short to pair the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Bispecific antibodies are more fully described in, for example, European Patent No. EP 404,097, International Patent Publication No. WO 93 / 11161, and Bolliger, et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0401] 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 that site. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation may increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in these cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, thereby resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. It also describes cell lines with reduced or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication No. WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740). International Patent Publication No. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, fucosidase enzymes 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.

[0402] "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).

[0403] 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 authorization or licensing purposes under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The original biological product already authorized is sometimes referred to as the "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on biosimilar medicinal products. Additionally, product-specific guidelines, including those related to monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are available on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy. 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 nonclinical studies. As used herein, the term "biosimilar" also refers to a biopharmaceutical that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may 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.

[0404] II. Gene Editing Process A. Overview: TIL expansion + gene editing Some embodiments of the present invention are directed to methods for expanding a TIL population (e.g., a TIL population expressing PD-1, CD39, and / or CD103). Some embodiments include one or more steps of gene editing at least a portion of the TILs to enhance their therapeutic efficacy. As used herein, "gene editing," "gene editing," and "genome editing" refer to types of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified, or replaced in the genome of a cell. In some embodiments, gene editing silences (sometimes referred to as gene knockout) or inhibits / reduces (sometimes referred to as gene knockdown) expression of a DNA sequence. In other embodiments, gene editing enhances expression of a DNA sequence (e.g., by causing overexpression). According to embodiments of the present invention, gene editing techniques are used to enhance the efficacy of therapeutic TIL populations.

[0405] In some embodiments, TIL populations are genetically modified to silence or reduce the expression of one or more cell surface receptors. In exemplary embodiments, the cell surface receptors are PD-1, CD39, and / or CD103. As used herein, "programmed cell death protein 1," "PD-1," "cluster of differentiation 279," and "CD279" all refer to a type I membrane protein expressed on immune cells (T cells and pro-B cells) that is a member of the extended CD28 / CTLA-4 family of T cell regulatory factors. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-1 and its ligands negatively regulate immune responses. For example, PD-L1 is highly expressed in some cancers, and inhibition of the PD-1 / PD-L1 interaction is thought to enhance T cell responses, thereby promoting anti-tumor activity. As used herein, "CD39," "ENTPD1," "ATPDase," "NTPDase-1," "SPG64," and "ectonucleoside triphosphohydrolase 1" all refer to cell surface enzymes that catalyze the hydrolysis of the γ- and β-phosphate residues of triphospho- and diphosphonucleosides to their monophosphonucleoside derivatives. High expression or activity of CD39 can prevent the immune system from inhibiting cancer progression. Indeed, both PD-1 and CD39 are commonly used markers of T cell exhaustion. As used herein, "CD103," "integrin subunit alpha E," "integrin, alpha E (antigen CD103, human mucosal lymphocyte antigen 1; alpha polypeptide)," "human mucosal lymphocyte antigen 1, alpha polypeptide," "mucosal lymphocyte 1 antigen," "integrin alpha IEL," "HUMINAE," "HML-1 antigen," and "CD103 antigen" all refer to an I-domain-containing alpha integrin that undergoes post-translational cleavage in the extracellular domain to yield disulfide-linked heavy and light chains and is preferentially expressed on lymphocytes. Enhanced expression of CD103 coincides with that of PD-1 and CD39, suggesting that this factor is phenotypically associated with these markers of T cell exhaustion and plays a role in the same phenotype observed with high expression of these other proteins.Therefore, without being bound by any particular theory of operation, just as PD-1 blockade increases anti-tumor activity in vivo, TILs genetically modified to silence or reduce expression of PD-1, CD39, and / or CD103 are believed to exhibit anti-tumor activity. In some embodiments, such TILs can evade PD-1-mediated checkpoint inhibition, and thus TILs can be modified to silence or reduce expression of CD39 and / or CD103 using any suitable method known in the art, including the genetic modification methods described herein. Exemplary genetic modification techniques include, for example, CRISPR, TALE, and zinc finger methods described herein.

[0406] In some embodiments, genetically modified TIL populations are first preselected for CD39 and / or CD103 expression, and the CD39- and / or CD103-expressing TIL populations are then genetically modified to silence or reduce PD-1, CD39, and / or CD103 expression. Without being bound by any particular theory of operation, it is believed that such TIL populations that have been genetically modified to silence or reduce PD-1, CD39, and CD103 expression will exhibit enhanced anti-tumor activity compared to a control TIL population (e.g., a TIL population that has not been preselected for CD39 and / or CD103 expression and / or that has been subsequently modified to reduce PD-1, CD39, and / or CD103 expression). TILs are preselected for CD39 and / or CD103 expression using any suitable method, including, for example, the CD39 and / or CD103 preselection methods provided herein.

[0407] In some embodiments, the genetically modified TIL population is expanded (after pre-selection for CD39 and / or CD103 expression and subsequent genetic modification to silence or reduce PD-1, CD39, and / or CD103 expression) to create a therapeutic TIL population genetically modified to silence or reduce PD-1, CD39, and / or CD103 expression. Any suitable expansion method can be used to expand the genetically modified TIL population, including the expansion methods provided herein.

[0408] A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein, which 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 embodiment of the methods described in U.S. Patent Application Publication No. 20180228841 A1 (U.S. Patent No. 10,517,894), U.S. Patent Application Publication No. 20200121719 A1, U.S. Patent Application Publication No. 20180282694 A1 (U.S. Patent No. 10,894,063), WO2020096986, WO2020096988, PCT / US21 / 30655 or U.S. Patent Application Publication No. 2021 / 0100842 A1 (all of which are incorporated by reference in their entireties), and the method further comprises gene editing at least a portion of the TILs. Thus, some embodiments of the invention provide therapeutic TIL populations pre-selected for CD39 and / or CD103 expression and expanded according to any embodiment described herein, wherein 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.

[0409] B. Timing of gene editing during TIL expansion In some embodiments, a population of TILs is genetically modified during the expansion methods provided herein. The expansion methods (e.g., the Gen2 and Gen3 processes described herein, or the process shown in FIG. 34) generally include a first expansion and a second expansion. In certain embodiments, the TILs are preselected for expression of CD39 and / or CD103 prior to the first expansion of the expansion method. In some embodiments, this CD39- and / or CD103-expressing population is genetically modified to silence or minimize expression of PD-1, CD39, and / or CD103 prior to undergoing the first expansion (e.g., the first expansion of the Gen2 and Gen3 processes described herein, or the first expansion shown in FIG. 34). In some embodiments, a population expressing CD39 and / or CD103 undergoes a first expansion, and the cells produced in the first expansion are genetically modified to silence or reduce expression of PD-1, CD39, and CD103 before undergoing a second expansion (e.g., a second expansion of the Gen2 and Gen3 processes described herein, or a second expansion as shown in FIG. 34). In some embodiments, a population expressing CD39 and / or CD103 undergoes a first and second expansion, and the TILs produced as a result of the second expansion are genetically modified to silence or reduce expansion of PD-1, CD39, and / or CD103.

[0410] According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining and / or receiving a first population of TILs in a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) selecting CD39 and / or CD103 positive TILs from the first TIL population of (a) to obtain a TIL population expressing CD39 and / or CD103; (c) performing a first priming expansion by culturing the CD39 and / or CD103-expressing TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 11 days to obtain a second TIL population, wherein the second TIL population produces a second TIL population that is greater in number than the first TIL population; (d) performing a second rapid expansion of the second TIL population by culturing the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of time of not more than about 14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population; (e) harvesting a therapeutic TIL population; (f) genetically modifying the first TIL population, the CD39 and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any point during the method after selection of CD39 and / or CD103 positive TILs from the first TIL population, such that the harvested therapeutic TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0411] As described in step (f) of the above embodiment, the genetic modification process may be performed on any TIL population in the present method, meaning that gene editing may be performed on the TILs before, during, or after any of the steps in the expansion method, for example, during any of steps (c)-(d) outlined in the above method. According to certain embodiments, the TILs are collected during the expansion method, and the collected TILs are subjected to the gene editing process, and in some cases, then returned to the expansion method (e.g., returned to culture medium) to continue the expansion process, thereby permanently gene editing at least a portion of the therapeutic TIL population. In some embodiments, the gene editing process may be performed prior to expansion by activating the TILs, performing a gene editing step on the activated TILs, and expanding the gene-edited TILs according to the processes described herein.

[0412] It should be noted that alternative embodiments of the expansion process may differ from the method shown above; for example, alternative embodiments may not have the same steps (a) to (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 expansions, and a gene modification step may be performed on the TIL during the third or fourth expansion, etc.

[0413] According to some embodiments, the genetic modification process is performed on TILs from one or more of the CD39 and / or CD103-expressing TILs, the second TIL population, and the third TIL population. For example, gene editing may be performed on the CD39 and / or CD103-expressing TIL population or on a portion of the TILs collected from the CD39 and / or CD103-expressing TIL population, and following the genetic editing process, those TILs may then be returned to the expansion process (e.g., returned to culture medium). Alternatively, genetic modification may be performed on TILs from the second or third population, or on a portion of the TILs collected from the second or third population, respectively, and following the genetic modification process, those TILs may then be returned to the expansion process (e.g., returned to culture medium). According to other embodiments, the genetic modification is performed while the TILs are still in culture medium and while expansion is taking place, i.e., they are not necessarily "removed" from the expansion to perform the genetic editing.

[0414] According to other embodiments, the genetic modification process is performed on TILs from the first expansion, or on TILs from the second expansion, or both. For example, during the first expansion or second expansion, genetic modification can be performed on TILs collected from the culture medium, and after the gene editing process, those TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium.

[0415] According to other embodiments, a genetic modification process is performed on at least a portion of the TILs after the first expansion and before the second expansion. For example, after the first expansion, gene editing can be performed on TILs collected from the culture medium, and after the genetic modification process, those TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium for the second expansion.

[0416] According to alternative embodiments, the gene editing process is carried out before step (c), before step (d), or before step (e).

[0417] In other embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments obtained from a tumor sample resected from a patient; (b) selecting CD39 and / or CD103 positive TILs from the first TIL population of (a) to obtain a TIL population expressing CD39 and / or CD103; (c) adding a population of TILs expressing CD39 and / or CD103 to the closed system; (d) performing a first expansion by culturing the TIL population expressing CD39 and / or CD103 in a cell culture medium comprising IL-2 and optionally OKT-3 (e.g., OKT-3 can be present in the cell culture medium starting on the initiation day of the expansion process) to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; and (g) transferring the harvested TIL population from step (f) into an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) genetically modifying the first TIL population, the CD39 and / or CD103-expressing TIL population, the second TIL population, and / or the third TIL population at any point during the method after selection of CD39 and / or CD103 positive TILs from the first TIL population, such that the harvested third TIL population comprises genetically modified TILs comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103.

[0418] As described in step (h) of the above embodiment, the genetic modification process may be carried out at any point during the TIL expansion method after selection of PD-1-positive TILs from the first TIL population and before transfer to the infusion bag in step (g). According to certain embodiments, the TILs are collected during the expansion method (e.g., the expansion method is "paused" for at least a portion of the TILs), the collected TILs are subjected to the gene editing process, and optionally then returned to the expansion method (e.g., returned to culture medium) to continue the expansion process, thereby permanently gene editing at least a portion of the therapeutic TIL population that will ultimately be transferred to the infusion bag. In some embodiments, the gene editing process may be carried out prior to expansion by activating the TILs, performing a gene editing step on the activated TILs, and expanding the gene-edited TILs according to the processes described herein.

[0419] It should be noted that alternative embodiments of the expansion process may differ from the method shown above; for example, alternative embodiments may not have the same steps (a) to (h) 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 after selecting PD-1-positive TILs from the first TIL population. For example, alternative embodiments may include more than two expansions, and gene editing may be performed on TILs during the third or fourth expansion, etc.

[0420] According to some embodiments, the gene editing process is performed on TILs from one or more of the CD39 and / or CD103-expressing TILs, the second TIL population, and the third TIL population. For example, gene editing may be performed on the CD39 and / or CD103-expressing TIL population or on a portion of TILs collected from the CD39 and / or CD103-expressing TIL population, and following the gene editing process, those TILs may then be returned to the expansion process (e.g., returned to culture medium). Alternatively, gene editing may be performed on TILs from the second or third population, or on a portion of TILs collected from the second or third population, respectively, and following the gene editing process, those TILs may then be returned to the expansion process (e.g., returned to culture medium). According to other embodiments, gene editing is performed while the TILs are still in culture medium and while expansion is being performed, i.e., they are not necessarily "removed" from the expansion to perform the gene editing.

[0421] According to other embodiments, the gene editing process is performed on TILs from the first expansion, or TILs from the second expansion, or both. For example, during the first expansion or second expansion, gene editing can be performed on TILs collected from the culture medium, and after the gene editing process, those TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium.

[0422] According to other embodiments, a gene editing process is performed on at least a portion of the TILs after the first expansion and before the second expansion. For example, after the first expansion, gene editing can be performed on TILs collected from the culture medium, and after the gene editing process, those TILs can then be returned to the expansion method, for example, by reintroducing them into the culture medium for the second expansion.

[0423] According to alternative embodiments, the gene editing process is carried out before step (d), before step (e), before step (f), or before step (g).

[0424] With respect to OKT-3, according to certain embodiments, the cell culture medium may include OKT-3 starting on the initiation day (day 0) or day 1 of the first expansion, such that gene editing is performed on the TILs after exposure to OKT-3 in the cell culture medium on day 0 and / or day 1. According to other embodiments, the cell culture medium includes OKT-3 during the first expansion and / or second expansion, and gene editing is performed before OKT-3 is introduced into the cell culture medium. Alternatively, the cell culture medium includes OKT-3 during the first expansion and / or second expansion, and gene editing is performed after OKT-3 is introduced into the cell culture medium.

[0425] With regard to the 4-1BB agonist, it should also be noted that, according to certain embodiments, the cell culture medium may include the 4-1BB agonist starting on the starting day (day 0) or day 1 of the first expansion, such that gene editing is performed on the TILs after exposure to the 4-1BB agonist in the cell culture medium on day 0 and / or day 1. According to other embodiments, the cell culture medium includes the 4-1BB agonist during the first expansion and / or second expansion, and gene editing is performed before the 4-1BB agonist is introduced into the cell culture medium. Alternatively, the cell culture medium includes the 4-1BB agonist during the first expansion and / or second expansion, and gene editing is performed after the 4-1BB agonist is introduced into the cell culture medium.

[0426] It is also noted that with respect to IL-2, according to certain embodiments, the cell culture medium may include IL-2 starting on the initiation day (day 0) or day 1 of the first expansion, such that gene editing is performed on the TILs after exposure to IL-2 in the cell culture medium on day 0 and / or day 1. According to other embodiments, the cell culture medium includes IL-2 during the first expansion and / or second expansion, and gene editing is performed before IL-2 is introduced into the cell culture medium. Alternatively, the cell culture medium includes IL-2 during the first expansion and / or second expansion, and gene editing is performed after IL-2 is introduced into the cell culture medium.

[0427] As described above, one or more of OKT-3, a 4-1BB agonist, and IL-2 may be included in the cell culture medium starting on day 0 or day 1 of the first expansion. According to some embodiments, OKT-3 is included in the cell culture medium starting on day 0 or day 1 of the first expansion, and / or a 4-1BB agonist is included in the cell culture medium starting on day 0 or day 1 of the first expansion, and / or IL-2 is included in the cell culture medium starting on day 0 or day 1 of the first expansion. According to one example, the cell culture medium includes OKT-3 and a 4-1BB agonist starting on day 0 or day 1 of the first expansion. According to another example, the cell culture medium includes OKT-3, a 4-1BB agonist, and IL-2 starting on day 0 or day 1 of the first expansion. Of course, as described in various embodiments herein, one or more of OKT-3, a 4-1BB agonist, and IL-2 may be added to the cell culture medium at one or more additional time points during the expansion process.

[0428] According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs from a tumor excised from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments; (b) selecting CD39 and / or CD103 positive TILs from the first TIL population of (a) to obtain a TIL population expressing CD39 and / or CD103; (c) adding a population of TILs expressing CD39 and / or CD103 to the closed system; (d) performing a first expansion by culturing the TILs expressing CD39 and / or CD103 in a cell culture medium containing IL-2, and optionally an OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (e) stimulating a second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (d) to step (e) occurs without opening the system; (f) sterile electroporating the second TIL population to transfer at least one gene editor into a portion of the cells of the second TIL population; (g) allowing the second population of TILs to rest for about 1 day; (h) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (g) to step (h) occurs without opening the system. (i) harvesting the therapeutic TIL population obtained from step (h) to provide a harvested TIL population, wherein the transition from step (h) to step (i) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (j) transferring the harvested TIL population into an infusion bag, wherein the transition from step (i) to (j) occurs without opening the system; Sterile electroporation of at least one gene editor into a portion of the cells of the second TIL population modifies that portion, or a plurality of cells in a third TIL population expanded from a portion of such TILs, to include a genetic modification that silences or reduces expression of endogenous PD-1, CD39, and / or CD103.

[0429] According to some embodiments, the method further comprises cryopreserving the collected TIL population using a cryopreservation medium. In some embodiments, the cryopreservation medium is a dimethyl sulfoxide-based cryopreservation medium. In other embodiments, the cryopreservation medium is CS10.

[0430] In other embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining and / or receiving a first population of TILs in a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) selecting CD39 and / or CD103 positive TILs from the first TIL population of (a) to obtain a TIL population expressing CD39 and / or CD103; (c) performing a first priming expansion of the PD-1-expressing TIL population by culturing it in a cell culture medium comprising IL-2, an anti-CD3 agonist antibody (e.g., OKT-3), and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of time of up to about 14 days to obtain a second TIL population, wherein the second TIL population produces a second TIL population that is greater in number than the first TIL population; (d) restimulating the second TIL population with an anti-CD3 agonist antibody (e.g., OKT-3); and (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces expression of PD-1, CD39, and / or CD103; (f) performing a second rapid expansion of the modified second TIL population by culturing the second TIL population in a second culture medium comprising IL-2, an anti-CD3 agonist antibody (e.g., OKT-3), and APCs to produce a third TIL population, wherein the second rapid expansion is performed for a second period of time of up to about 14 days to obtain a third TIL population, wherein the third TIL population is a therapeutic TIL population comprising a genetic modification that reduces expression of PD-1, CD39, and / or CD103; (g) harvesting a third population of TILs.

[0431] In some embodiments, the first expansion by priming is carried out for a first period of about 5 days, about 7 days, or about 11 days.

[0432] In some embodiments, the second TIL population is restimulated for about 2 days. In some embodiments, the anti-CD3 agonist antibody used for restimulation is part of an anti-CD3 / anti-CD28 antibody bead. In other embodiments, the anti-CD3 antibody is OKT-3.

[0433] In some embodiments, the rapid secondary expansion is carried out over a period of about 7-11 days. In some embodiments, the rapid secondary expansion includes culture splitting and scale-up about 5 days after the rapid secondary expansion. In such embodiments, the subculture is seeded into a new flask with fresh medium and IL-2 and cultured for about 6 more days.

[0434] In some embodiments, the genetic modification step comprises electroporation and delivery of at least one gene editor system selected from the group consisting of a clustered regularly interspersed short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system reduces expression of PD-1, CD39, and / or CD103 in the modified second TIL population.

[0435] According to some embodiments, the methods described above can be used to provide autologous TIL populations for the treatment of human subjects with cancer.

[0436] C. Gene Editing Methods As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been genetically modified via gene editing to enhance their therapeutic effect (e.g., to silence or reduce expression of endogenous PD-1, CD39, and / or CD103). Embodiments of the present invention encompass gene editing by inserting nucleotides (RNA or DNA) into a TIL population to both promote expression of one or more proteins and inhibit expression of one or more proteins, as well as combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into a therapeutic population, the methods comprising genetically editing the TILs. There are several gene editing techniques that can be used to genetically modify a TIL population, which are suitable for use according to the present invention.

[0437] In some embodiments, the method of genetically modifying a TIL population comprises a step of stable integration of a gene for the production of one or more proteins. In some embodiments, the method of genetically modifying a TIL population comprises a step of retroviral transduction. In some embodiments, the method of genetically modifying a TIL population comprises a step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71; and U.S. Pat. No. 6,627,442 (the disclosures of each of which are incorporated herein by reference). In some embodiments, the method of genetically modifying a TIL population comprises a step of gammaretroviral transduction. Gammaretroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein, thereby preventing long-term expression of the transposase, e.g., provided as mRNA (e.g., mRNA comprising a cap and polyA tail), in the transgenic cells. Suitable transposon-mediated gene transfer systems, including salmonid-type Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, as well as engineered enzymes with increased enzymatic activity, are described, for example, in Hackett, et al., Mol. Therapy 2010, 18,674-83 and U.S. Patent No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

[0438] In some embodiments, the method of genetically modifying a TIL population comprises a step of stable integration of a gene for the production or inhibition (e.g., silencing) of one or more proteins. In some embodiments, the method of genetically modifying a TIL population comprises a step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014 / 0227237A1, the disclosures of each of which are incorporated herein by reference. Other electroporation methods known in the art can be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490 (the disclosures of which are incorporated herein by reference). In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulse electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a defined and controlled permanent or temporary alteration of the TIL, and comprising applying to the TIL at least three single-operator-controlled, independently programmed DC electric pulse trains having a field strength of 100 V / cm or greater, wherein the at least three DC electric pulse trains have one, two, or three of the following characteristics: (1) at least two of the at least three pulses have pulse amplitudes that are different from each other; (2) at least two of the at least three pulses have pulse widths that are different from each other; and (3) a first pulse interval of a first set of two of the at least three pulses is different from a second pulse interval of a second set of two of the at least three pulses.In some embodiments, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a controlled, permanent or temporary alteration of the TIL's definition, comprising applying to the TIL at least three single-operator-controlled, independently programmed DC electrical pulse trains having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses have different pulse amplitudes. In some embodiments, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a controlled, permanent or temporary alteration of the TIL's definition, comprising applying to the TIL at least three single-operator-controlled, independently programmed DC electrical pulse trains having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses have different pulse widths. In some embodiments, the electroporation method is a pulsed electroporation method comprising treating TILs with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary alteration of the TILs, comprising applying to the TILs at least three single-operator-controlled, independently programmed DC electrical pulse trains having a field strength of 100 V / cm or greater, wherein a first pulse interval of at least two of the at least three pulses is different from a second pulse interval of two of the at least three pulses.In some embodiments, the electroporation method is a pulsed electroporation method comprising treating TILs with a pulsed electric field to induce pore formation in the TILs, comprising applying to the TILs a train of at least three DC electric pulses having a field strength of 100 V / cm or greater, wherein the train of at least three DC electric pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses have pulse amplitudes that differ from each other; (2) at least two of the at least three pulses have pulse widths that differ from each other; and (3) a first pulse interval of a first set of two of the at least three pulses differs from a second pulse interval of a second set of two of the at least three pulses, thereby sustaining the induced pores for a relatively long period of time and maintaining the viability of the TILs. In some embodiments, the method for genetically modifying a TIL population comprises a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752, and U.S. Patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population includes a liposome transfection step.Liposomal transfection methods, such as those using a 1:1 (w / w) liposomal formulation of the cationic lipids N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphotidylethanolamine (DOPE), are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al. al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patent Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises a transfection step using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of each of which are incorporated herein by reference.

[0439] According to certain embodiments, the gene editing process can involve the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci; that is, they rely on the recognition of specific DNA sequences within the genome to target the nuclease domain to this position and mediate the generation of double-stranded breaks at the target sequence. The double-stranded break in DNA then recruits endogenous repair mechanisms to the break site to mediate genome editing by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). Therefore, repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, suppress, or enhance) the target gene product.

[0440] The major classes of nucleases developed to enable site-specific genome editing include zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR / Cas9). These nuclease systems can be broadly classified into two categories based on the mode of DNA recognition: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, while CRISPR systems such as Cas9 target specific DNA sequences through short RNA guide molecules that directly base pair with the target DNA and through protein-DNA interactions. See, for example, Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

[0441] Non-limiting examples of gene editing methods that can be used according to the TIL expansion methods of the present invention include CRISPR, TALE, and ZFN methods, embodiments of which are described in more detail below. According to some embodiments, the method for expanding TILs into a therapeutic population can be carried out according to any embodiment of the method described herein (e.g., Process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, and the method further includes gene editing at least a portion of the TILs using one or more of CRISPR, TALE, or ZFN methods to generate TILs that can provide an enhanced therapeutic effect. According to some embodiments, the gene-edited TILs can be evaluated for improved therapeutic effect by comparing them to unmodified TILs in vitro, for example, by evaluating in vitro effector function, cytokine profile, etc., compared to unmodified TILs.

[0442] In some embodiments of the present invention, electroporation is used to deliver gene editing systems such as CRISPR systems, TALEN systems, and ZFN systems. In some embodiments of the present invention, the electroporation system is a flow electroporation system. An example of a suitable flow electroporation system suitable for use with some embodiments of the present invention is the commercially available MaxCyte STX system. There are several alternative commercially available electroporation devices that may be suitable for use with the present invention, such as the AgilePulse system or ECM830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza / Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax), or siPORTer96 (Ambion). In some embodiments of the present invention, the electroporation system, together with the rest of the TIL expansion method, forms a sterile, closed system. In some embodiments of the present invention, the electroporation system is a pulse electroporation system as described herein, and, together with the rest of the TIL expansion method, forms a sterile, closed system.

[0443] D. Immune Checkpoints According to certain embodiments of the invention, a TIL population (i.e., a TIL population enriched for CD39 and / or CD103 expression) is gene edited to silence or reduce expression of one or more immune checkpoint genes. In an exemplary embodiment, the immune checkpoint gene is PD-1.

[0444] Immune checkpoints are molecules expressed by lymphocytes that regulate immune responses through inhibitory or stimulatory pathways. In cancer, immune checkpoint pathways are often activated to inhibit anti-tumor responses; that is, the expression of certain immune checkpoints by malignant cells inhibits anti-tumor immunity and promotes the growth of cancer cells. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39. Therefore, certain inhibitory checkpoint molecules serve as targets for immunotherapy in the present invention. According to certain embodiments, TILs are gene-edited to block or stimulate certain immune checkpoint pathways, thereby enhancing the body's immunological activity against tumors.

[0445] As used herein, immune checkpoint genes include DNA sequences that encode immune checkpoint molecules. According to certain embodiments of the present invention, gene-edited TILs in the TIL expansion method silence or reduce the expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. For example, gene editing can silence or reduce the expression of inhibitory receptors such as PD-1 or CTLA-4 to enhance immune response.

[0446] The most widely studied checkpoints include programmed death receptor-1 (PD-1) and cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4), which are inhibitory receptors on immune cells that inhibit key effector functions (e.g., activation, proliferation, cytokine release, cytotoxicity, etc.) when they interact with inhibitory ligands. In addition to PD-1 and CTLA-4, numerous checkpoint molecules have emerged as potential targets for immunotherapy, as discussed in more detail below.

[0447] Non-limiting examples of immune checkpoint genes that can be silenced or inhibited by permanently gene editing the TILs of the present invention include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10 Immune checkpoint genes that can be silenced or inhibited in the TILs of the present invention include B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that can be silenced or inhibited in the TILs of the present invention may be selected from the group including PD-1, CTLA-4, LAG-3, TIM-3, Cish, TGFβ, and PKA. BAFF (BR3) is described in Bloom et al., J. Immunother., 2018, in press. According to another embodiment, the immune checkpoint genes that may be silenced or inhibited in the TILs of the present invention may be selected from the group including PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0448] According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments generated from a tumor sample resected from a patient; (b) selecting CD39 and / or CD103 positive TILs from the first TIL population of (a) to obtain a TIL population expressing CD39 and / or CD103; (c) adding a population of TILs expressing CD39 and / or CD103 to the closed system; (d) performing a first expansion by culturing the TIL population expressing CD39 and / or CD103 in cell culture medium containing IL-2, and optionally containing OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (e) stimulating a second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (f) sterile electroporating the second TIL population to transfer at least one gene editor into a plurality of cells of the second TIL population, wherein the transition from step (e) to step (f) occurs without opening the system; and (g) allowing the second TIL population to rest for about 1 day, wherein the transition from step (f) to step (g) occurs without opening the system; (h) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (g) to step (h) occurs without opening the system; (i) harvesting the third TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (h) to step (i) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (j) transferring the harvested TIL population into an infusion bag, wherein the transition from step (i) to (j) occurs without opening the system; (k) optionally, cryopreserving the harvested TIL population using a cryopreservation medium; The electroporation step comprises delivery of at least one gene editor system selected from the group consisting of a clustered regularly interspersed short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system. In some embodiments, the effect of the at least one gene editor system inhibits expression of one or more molecules selected from the group consisting of PD-1, CD39 and / or CD103, and LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3) in a plurality of cells of the second TIL population.

[0449] 1.PD-1 One of the most studied targets for inducing checkpoint blockade is the programmed death receptor (PD1 or PD-1, also known as PDCD1), a member of the CD28 superfamily of T cell regulatory factors. Its ligands, PD-L1 and PD-L2, are expressed on a variety of tumor cells, including melanoma. The interaction of PD-1 with PD-L1 inhibits T cell effector function, leads to T cell exhaustion in the setting of chronic stimulation, and induces T cell apoptosis in the tumor microenvironment. PD1 may also play a role in tumor-specific escape from immune surveillance.

[0450] According to certain embodiments, the expression of PD1 in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the method described herein (e.g., Process 2A, Process Gen3, or the method shown in Figure 34), which method includes gene editing at least a portion of the TILs by silencing or suppressing the expression of PD1. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as PD1. For example, CRISPR, TALE, or zinc finger methods can be used to silence or reduce the expression of PD1 in TILs.

[0451] 2.CTLA-4 CTLA-4 expression is induced on activated T cells during T cell activation and competes for binding with antigen-presenting cell activation antigens CD80 and CD86. The interaction of CTLA-4 with CD80 or CD86 causes T cell inhibition and helps maintain the balance of the immune response. However, inhibition of CTLA-4 interaction with CD80 or CD86 can prolong T cell activation and thus increase the level of immune response to cancer antigens.

[0452] According to certain embodiments, the expression of CTLA-4 in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the methods described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs to silence or suppress the expression of CTLA-4 in the TILs. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as CTLA-4. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of CTLA-4 in TILs.

[0453] 3. LAG-3 Lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells and natural killer (NK) cells after major histocompatibility complex (MHC) class II ligation. Although the mechanism remains unclear, its regulation causes a negative regulatory effect on T cell function, preventing tissue damage and autoimmunity. LAG-3 and PD-1 are often coexpressed and upregulated on TILs, leading to immune exhaustion and tumor growth. Therefore, LAG-3 blockade improves antitumor responses. See, for example, Marin-Acevedo et al., Journal of Hematology & Oncology (2018) 11:39.

[0454] According to certain embodiments, the expression of LAG-3 in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the methods described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs to silence or suppress LAG-3 expression in the TILs. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as LAG-3. According to certain embodiments, CRISPR, TALE, or zinc finger methods can be used to silence or suppress LAG-3 expression in TILs.

[0455] 4. TIM-3 T cell immunoglobulin-3 (TIM-3) is a direct negative regulator of T cells and is expressed on NK cells and macrophages. TIM-3 indirectly promotes immunosuppression by inducing the proliferation of myeloid-derived suppressor cells (MDSCs). Its levels have been found to be particularly elevated in dysfunctional and exhausted T cells, suggesting an important role in malignancies.

[0456] According to certain embodiments, the expression of TIM-3 in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the methods described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs to silence or suppress the expression of TIM-3 in the TILs. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as TIM-3. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of TIM-3 in TILs.

[0457] 5. Cish Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional inactivation against tumors. Genetic deletion of Cish in CD8+ T cells can enhance their expansion, functional affinity, and cytokine multifunction, resulting in significant and durable regression of established tumors. See, e.g., Palmer et al., Journal of Experimental Medicine, 212(12):2095 (2015).

[0458] According to certain embodiments, expression of Cish in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the method described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs to silence or suppress expression of Cish in the TILs. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as Cish. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress expression of Cish in TILs.

[0459] 6. TGFβ The TGFβ signaling pathway has multiple functions in regulating cell proliferation, differentiation, apoptosis, motility and invasion, extracellular matrix production, angiogenesis, and immune response. Deregulation of TGFβ signaling frequently occurs in tumors and plays an important role in tumor initiation, development, and metastasis. At the microenvironment level, the TGFβ pathway contributes to generating a favorable microenvironment for tumor growth and metastasis throughout carcinogenesis. See, for example, Neuzillet et al., Pharmacology & Therapeutics, Vol. 147, pp. 22-31 (2015).

[0460] According to certain embodiments, the expression of TGFβ in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the method described herein (e.g., Process 2A, Process Gen3, or the method shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs to silence or reduce the expression of TGFβ in the TILs. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as TGFβ. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of TGFβ in TILs.

[0461] In some embodiments, TGFβR2 (TGF beta receptor 2) can be inhibited by silencing TGFβR2 using the CRISPR / Cas9 system or by using a TGFβR2 dominant-negative extracellular trap using methods known in the art.

[0462] 7. PKA Protein kinase A (PKA) is a well-known member of the serine-threonine protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multiunit protein kinase that mediates signal transduction of G protein-coupled receptors via activation upon cAMP binding. It is involved in the regulation of a wide variety of cellular processes, from metabolism to ion channel activation, cell growth and differentiation, gene expression, and apoptosis. Importantly, PKA is involved in the initiation and progression of many tumors. See, e.g., Sapio et al., EXCLI Journal; 2014; 13:843-855.

[0463] According to certain embodiments, the expression of PKA in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the methods described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), which method comprises gene editing at least a portion of the TILs to silence or suppress the expression of PKA in the TILs. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as PKA. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of PKA in TILs.

[0464] 8.CBLB CBLB (or CBL-B) is an E3 ubiquitin-protein ligase and a negative regulator of T cell activation. Bachmaier, et al., Nature, 2000, 403, 211-216; Wallner, et al., Clin. Dev. Immunol. 2012, 692-639.

[0465] According to certain embodiments, the expression of CBLB in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the methods described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs to silence or suppress the expression of CBLB in the TILs. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double- or single-strand breaks in immune checkpoint genes, such as CBLB. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of PKA in TILs. In some embodiments, CBLB is silenced using TALEN knockout. In some embodiments, CBLB is silenced using TALE-KRAB transcription inhibitor knockin. Further details of these methods can be found in Boettcher and McManus, Mol. Cell Review, 2015, 58, 575-585.

[0466] 9. TIGIT T cell immunoreceptors with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains, or TIGIT, are transmembrane glycoprotein receptors with an Ig-like V-type domain and an ITIM in their cytoplasmic domain. Khalil, et al., Advances in Cancer Research, 2015, 128, 1-68; Yu, et al., Nature Immunology, 2009, Vol. 10, No. 1, 48-57. TIGIT is expressed by some T cells and natural killer cells. Furthermore, TIGIT has been shown to be overexpressed on antigen-specific CD8+ T cells and CD8+ TILs, particularly from individuals with melanoma. Studies have shown that the TIGIT pathway contributes to tumor immune evasion and that TIGIT inhibition increases T cell activation and proliferation in response to polyclonal and antigen-specific stimulation. Khalil, et al., Advances in Cancer Research, 2015, 128, 1-68. Furthermore, co-blockade of TIGIT with either PD-1 or TIM3 has shown synergistic effects against solid tumors in mouse models. Id.; See also Kurtulus, et al., The Journal of Clinical Investigation, 2015, Vol. 125, No. 11, 4053-4062.

[0467] According to certain embodiments, the expression of TIGIT in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the method described herein (e.g., Process 2A, Process Gen3, or the method shown in Figures 34 and 35), which method comprises gene editing at least a portion of the TILs to silence or suppress the expression of TIGIT in the TILs. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks in immune checkpoint genes such as TIGIT. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress the expression of TIGIT in TILs.

[0468] 10. TOX Thymocyte selection-associated high-mobility group (HMG) box (TOX) is a transcription factor containing an HMG box DNA-binding domain. TOX is a member of the HMG box superfamily that appears to bind DNA in a sequence-independent but structure-dependent manner.

[0469] TOX expresses tumor-specific CD8 + CD8 T cells have been identified as important regulators of T cell dysfunction or T cell exhaustion, as described, for example, in Scott, et al., Nature, 2019, 571, 270-274 and Khan, et al., Nature, 2019, 571, 211-218 (both of which are incorporated by reference in their entirety). +TOX has been found to transcriptionally and epigenetically program T cell exhaustion. TOX has also been found to be a key factor in the progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, as described in Alfei, et al., Nature, 2019, 571, 265-269 (incorporated herein by reference in its entirety). TOX is highly expressed in dysfunctional or exhausted T cells from tumors and chronic viral infections. Ectopic expression of TOX in effector T cells in vitro induced the transcriptional program associated with T cell exhaustion, whereas deletion of TOX in T cells abolished the T exhaustion program.

[0470] According to certain embodiments, the expression of TOX in TILs is silenced or reduced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any of the methods described herein (e.g., Process 2A, Process GEN3, or the methods shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs to silence or suppress TOX expression. As described in more detail below, the gene editing process can include the use of a programmable nuclease that mediates the generation of double- or single-strand breaks in immune checkpoint genes such as TOX. For example, CRISPR, TALE, or zinc finger methods can be used to silence or suppress TOX expression in TILs.

[0471] E. Overexpression of costimulatory receptors or adhesion molecules According to additional embodiments, the gene-edited TILs in the TIL expansion method enhance the expression of one or more costimulatory receptors, adhesion molecules, and / or cytokines in at least a portion of the therapeutic TIL population. For example, gene editing can enhance the expression of a costimulatory receptor, adhesion molecule, or cytokine, meaning that it is overexpressed compared to the expression of a costimulatory receptor, adhesion molecule, or cytokine that is not genetically modified. Non-limiting examples of costimulatory receptor, adhesion molecule, or cytokine genes that may exhibit enhanced expression by permanently gene editing the TILs of the present invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH1 / 2 intracellular domain (ICD), and / or the NOTCH ligand mDLL1.

[0472] 1. CCR For adoptive T cell immunotherapy to be effective, T cells must be appropriately trafficked into tumors by chemokines. The correspondence between chemokines secreted by tumor cells, chemokines present in the periphery, and chemokine receptors expressed by T cells is crucial for successful trafficking of T cells into the tumor bed.

[0473] According to certain embodiments, the gene editing methods of the present invention may be used to increase the expression of certain chemokine receptors in TILs, such as one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1. Overexpression of CCRs can help promote effector function and proliferation of TILs after adoptive transfer.

[0474] According to certain embodiments, expression in TILs of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1 is enhanced according to the compositions and methods of the invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be carried out according to any embodiment of the methods described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), comprising gene editing at least a portion of the TILs to express and enhance expression of at least one immunomodulatory composition on the cell surface of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and CX3CR1 in the TILs.

[0475] As will be described in more detail below, gene editing process can include using programmable nucleases to mediate the generation of double-stranded or single-stranded breaks in chemokine receptor genes.For example, CRISPR, TALE or zinc finger methods can be used to enhance the expression of certain chemokine receptors in TILs.

[0476] In some embodiments, CCR4 and / or CCR5 adhesion molecules are inserted into TIL populations using gamma-retroviral or lentiviral methods described herein. In some embodiments, CXCR2 adhesion molecules are inserted into TIL populations using gamma-retroviral or lentiviral methods such as those described in Forget, et al., Frontiers Immunology 2017, 8, 908 or Peng, et al., Clin. Cancer Res. 2010, 16, 5458, the disclosures of which are incorporated herein by reference.

[0477] According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments generated from a tumor sample resected from a patient; (b) adding multiple tumor fragments to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (d) stimulating the second TIL population by adding OKT-3 and culturing for about 1 to 3 days to obtain a second TIL population, wherein the transition from step (c) to step (d) occurs without opening the system; and (e) sterile electroporating the second TIL population to transfer at least one gene editor into a plurality of cells of the second TIL population, wherein the transition from step (d) to step (e) occurs without opening the system; and (f) allowing the second TIL population to rest for about 1 day, wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) optionally, cryopreserving the harvested TIL population using a cryopreservation medium; The electroporation step includes delivery of at least one gene editor system selected from the group consisting of a clustered regularly interspersed short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system inhibits expression of PD-1 and optionally LAG-3 in a plurality of cells of the second TIL population, and wherein the at least one gene editor system expresses a CXCR2 adhesion molecule on the cell surface of a plurality of cells of the second TIL population, or the CXCR2 adhesion molecule is inserted into the first TIL population, the second TIL population, or the harvested TIL population by gammaretroviral or lentiviral methods.

[0478] According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments generated from a tumor sample resected from a patient; (b) adding multiple tumor fragments to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (d) stimulating a second TIL population by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) occurs without opening the system; (e) sterile electroporating the second TIL population to transfer at least one gene editor into a plurality of cells of the second TIL population, wherein the transition from step (d) to step (e) occurs without opening the system; and (f) allowing the second TIL population to rest for about 1 day, wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) optionally, cryopreserving the harvested TIL population using a cryopreservation medium; The electroporation step includes delivery of at least one gene editor system selected from the group consisting of a clustered regularly interspersed short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system inhibits expression of PD-1 and optionally LAG-3 in a plurality of cells of the second TIL population, and wherein the at least one gene editor system expresses CCR4 and / or CCR5 adhesion molecules on the cell surface of a plurality of cells of the second TIL population, or the CCR4 and / or CCR5 adhesion molecules are inserted into the first TIL population, the second TIL population, or the harvested TIL population by gammaretroviral or lentiviral methods.

[0479] According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments generated from a tumor sample resected from a patient; (b) adding multiple tumor fragments to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (d) stimulating the second TIL population by adding OKT-3 and culturing for about 1 to 3 days to obtain a second TIL population, wherein the transition from step (c) to step (d) occurs without opening the system; and (e) sterile electroporating the second TIL population to transfer at least one gene editor into a plurality of cells of the second TIL population, wherein the transition from step (d) to step (e) occurs without opening the system; and (f) allowing the second TIL population to rest for about 1 day, wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the third TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) optionally, cryopreserving the harvested TIL population using a cryopreservation medium; The electroporation step includes delivery of at least one gene editor system selected from the group consisting of a clustered regularly interspersed short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system inhibits expression of PD-1 and optionally LAG-3 in a plurality of cells of the second TIL population, and wherein the at least one gene editor system expresses an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof on the cell surface of a plurality of cells of the second TIL population, or the adhesion molecule is inserted into the first TIL population, the second TIL population, or the harvested TIL population by gammaretroviral or lentiviral methods.

[0480] 2. Interleukins According to additional embodiments, the gene editing methods of the invention may be used to increase the expression of certain interleukins, such as one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21. Certain interleukins have been demonstrated to increase T cell effector function and mediate tumor control.

[0481] According to certain embodiments, the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21 in TILs is enhanced according to the compositions and methods of the present invention. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any of the methods described herein (e.g., Process 2A, Process Gen3, or the methods shown in Figures 34 and 35), which method includes gene editing at least a portion of the TILs by enhancing the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21. As described in more detail below, the gene editing process can include the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in interleukin genes. For example, CRISPR, TALE, or zinc finger methods can be used to enhance the expression of certain interleukins in TILs.

[0482] According to some embodiments, the method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population comprises: (a) obtaining a first population of TILs in a plurality of tumor fragments produced from a tumor resected from a patient; (b) adding multiple tumor fragments to a closed system; (c) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2, and optionally containing an OKT-3 and / or 4-1BB agonist antibody, for about 3 to 11 days to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area; (d) stimulating the second TIL population by adding OKT-3 and culturing for about 1 to 3 days to obtain a second TIL population, wherein the transition from step (c) to step (d) occurs without opening the system; and (e) sterile electroporating the second population of TILs to transfer at least one gene editor, wherein the transfer from step (d) to step (e) occurs without opening the system; and (f) allowing the second TIL population to settle within the plurality of cells of the second TIL population for about 1 day, wherein the transition from step (e) to step (f) occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally OKT-3 antibody, optionally OX40 antibody, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days, and the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) occurs without opening the system; (h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population, wherein the transition from step (g) to step (h) occurs without opening the system, and wherein the harvested TIL population is a therapeutic TIL population; (i) transferring the harvested TIL population into an infusion bag, wherein the transition from step (h) to (i) occurs without opening the system; (j) optionally, cryopreserving the harvested TIL population using a cryopreservation medium; The electroporation step includes delivery of at least one gene editor system selected from the group consisting of a clustered regularly interspersed short palindromic repeats (CRISPR) system, a transcription activator-like effector (TALE) system, or a zinc finger system, wherein the at least one gene editor system inhibits expression of PD-1 and optionally LAG-3 in a plurality of cells of the second TIL population, and wherein the at least one gene editor system expresses an interleukin selected from the group consisting of IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, and combinations thereof at the cell surface of a plurality of cells of the second TIL population, or the interleukin is inserted into the first TIL population, the second TIL population, or the harvested TIL population by gammaretroviral or lentiviral methods.

[0483] 3. Gene editing methods As discussed above, embodiments of the present invention provide tumor-infiltrating lymphocytes (TILs) that have been genetically modified via gene editing to enhance their therapeutic efficacy. Embodiments of the present invention encompass gene editing by inserting nucleotides (RNA or DNA) into a TIL population to both promote the expression of one or more proteins and inhibit the expression of one or more proteins, as well as combinations thereof. Embodiments of the present invention also provide methods for expanding TILs into a therapeutic population, which methods include genetically editing the TILs. There are several gene editing techniques that can be used to genetically modify a TIL population, and they are suitable for use in the present invention. In some embodiments, electroporation is used as part of the gene editing method.

[0484] In some embodiments, the method of genetically modifying a TIL population comprises a step of stable integration of a gene for the production of one or more proteins. In some embodiments, the method of genetically modifying a TIL population comprises a step of retroviral transduction. In some embodiments, the method of genetically modifying a TIL population comprises a step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, for example, in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71; and U.S. Pat. No. 6,627,442 (the disclosures of each of which are incorporated herein by reference). In some embodiments, the method of genetically modifying a TIL population comprises a step of gammaretroviral transduction. Gammaretroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided as a DNA expression vector or as an expressible RNA or protein, thereby preventing long-term expression of the transposase, e.g., provided as mRNA (e.g., mRNA comprising a cap and polyA tail), in the transgenic cells. Suitable transposon-mediated gene transfer systems, including salmonid-type Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, as well as engineered enzymes with increased enzymatic activity, are described, for example, in Hackett, et al., Mol. Therapy 2010, 18,674-83 and U.S. Patent No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

[0485] In some embodiments, the method of genetically modifying a TIL population comprises a step of stable integration of a gene for the production or inhibition (e.g., silencing) of one or more proteins. In some embodiments, the method of genetically modifying a TIL population comprises a step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014 / 0227237 A1, the disclosures of each of which are incorporated herein by reference. Other electroporation methods known in the art can be used, such as those described in U.S. Patent Nos. 5,019,034, 5,128,257, 5,137,817, 5,173,158, 5,232,856, 5,273,525, 5,304,120, 5,318,514, 6,010,613, and 6,078,490 (the disclosures of which are incorporated herein by reference). In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulse electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a defined and controlled permanent or temporary alteration of the TIL, and comprising applying to the TIL at least three single-operator-controlled, independently programmed DC electric pulse trains having a field strength of 100 V / cm or greater, wherein the at least three DC electric pulse trains have one, two, or three of the following characteristics: (1) at least two of the at least three pulses have pulse amplitudes that are different from each other; (2) at least two of the at least three pulses have pulse widths that are different from each other; and (3) a first pulse interval of a first set of two of the at least three pulses is different from a second pulse interval of a second set of two of the at least three pulses.In some embodiments, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a controlled, permanent or temporary alteration of the TIL's definition, comprising applying to the TIL at least three single-operator-controlled, independently programmed DC electrical pulse trains having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses have different pulse amplitudes. In some embodiments, the electroporation method is a pulsed electroporation method comprising treating a TIL with a pulsed electric field to alter, manipulate, or cause a controlled, permanent or temporary alteration of the TIL's definition, comprising applying to the TIL at least three single-operator-controlled, independently programmed DC electrical pulse trains having a field strength of 100 V / cm or greater, wherein at least two of the at least three pulses have different pulse widths. In some embodiments, the electroporation method is a pulsed electroporation method comprising treating TILs with a pulsed electric field to alter, manipulate, or cause defined and controlled permanent or temporary alteration of the TILs, comprising applying to the TILs at least three single-operator-controlled, independently programmed DC electrical pulse trains having a field strength of 100 V / cm or greater, wherein a first pulse interval of at least two of the at least three pulses is different from a second pulse interval of two of the at least three pulses.In some embodiments, the electroporation method is a pulsed electroporation method comprising treating the TIL with a pulsed electric field to induce pore formation in the TIL, and comprising applying to the TIL a train of at least three DC electric pulses having an electric field strength of 100 V / cm or more, wherein the train of at least three DC electric pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses have pulse amplitudes that are different from each other; (2) at least two of the at least three pulses have pulse widths that are different from each other; and (3) a first pulse interval of a first set of two of the at least three pulses is different from a second pulse interval of a second set of two of the at least three pulses, thereby sustaining the induced pores for a relatively long period of time and maintaining the viability of the TIL.

[0486] In some embodiments, the method for genetically modifying a TIL population comprises a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752, and U.S. Patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method for genetically modifying a TIL population comprises a liposome transfection step. Liposomal transfection methods, such as those using a 1:1 (w / w) liposomal formulation of the cationic lipids N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphotidylethanolamine (DOPE), are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al. al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417, and U.S. Patent Nos. 5,279,833, 5,908,635, 6,056,938, 6,110,490, 6,534,484, and 7,687,070, the disclosures of each of which are incorporated herein by reference. In some embodiments, the method of genetically modifying a TIL population comprises a transfection step using the methods described in U.S. Patent Nos. 5,766,902, 6,025,337, 6,410,517, 6,475,994, and 7,189,705, the disclosures of each of which are incorporated herein by reference.

[0487] According to some embodiments, the gene editing process can involve the use of programmable nucleases that mediate the generation of double-stranded or single-stranded breaks in one or more immune checkpoint genes. Such prog...

Claims

1. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor infiltrating lymphocytes (TILs), said method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from the subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) optionally adding said TIL population to a closed system; (d) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is optionally performed in a sealed container that provides a second gas permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested third population of TILs from step (g) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the subject from the infusion bag of step (h); (j) optionally, genetically modifying the TIL population any time prior to the administering step (i) such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

2. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TILs), said method comprising: (a) obtaining a first population of TILs from a tumor excised from the subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) optionally adding said TIL population to a closed system; (d) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) optionally occurs without opening the system to produce the third TIL population; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the subject from the infusion bag of step (h); (j) optionally, genetically modifying the TIL population any time prior to the administering step (i) such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

3. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TILs), said method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer in the patient or subject; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) optionally adding said TIL population to a closed system; (d) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) optionally occurs without opening the system to produce the third TIL population; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (e) to (f) optionally occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the subject from the infusion bag of step (h); (j) optionally, genetically modifying the TIL population any time prior to the administering step (i) such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

4. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of modified tumor infiltrating lymphocytes (TILs), said method comprising: (a) resecting a tumor from said subject or patient, said tumor optionally comprising a first TIL population from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from said cancer; (b) processing the tumor into a plurality of tumor fragments; (c) enzymatically digesting the plurality of tumor fragments to obtain the first population of TILs; (d) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (e) optionally adding said TIL population to a closed system; (f) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) optionally occurs without opening the system to produce the third TIL population; (h) harvesting the third population of TILs obtained from step (g), wherein the transition from step (g) to step (h) optionally occurs without opening the system; (i) transferring the harvested third population of TILs from step (h) to an infusion bag, wherein the transition from step (h) to (i) optionally occurs without opening the system; (j) cryopreserving the infusion bag containing the harvested TIL population from step (i) using a cryopreservation process; (k) administering a therapeutically effective dose of the third population of TILs from the infusion bag of step (g) to the subject or patient with cancer; (j) optionally, genetically modifying the TIL population, the second TIL population, and / or the third TIL population at any time prior to the administering step (k), such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

5. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TILs), said method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from said subject or said patient; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) contacting the TIL population with a first cell culture medium; (d) performing a first expansion of the TIL population in the first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion occurs over a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after initiation of the rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APC, the rapid expansion being performed over a period of 14 days or less, and optionally, the rapid second expansion being allowed to proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion; (f) harvesting the third population of TILs; (g) administering a therapeutically effective portion of the third population of TILs to the subject or patient with the cancer; (h) optionally, genetically modifying the TIL population at any time prior to the administering step (g) such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

6. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TILs), said method comprising: (a) resecting a tumor from said cancer in said subject or patient, said tumor optionally comprising a first TIL population from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from said cancer; (b) fragmenting the tumor into tumor fragments or processing the tumor into a tumor digest; (c) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (d) contacting the tumor fragment with a first cell culture medium; (e) performing a first expansion of the TIL population in the first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion occurs over a period of 1 to 8 days; (f) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after initiation of the rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APC, the rapid expansion being performed over a period of 14 days or less, and optionally, the rapid second expansion being allowed to proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion; (g) harvesting the third population of TILs; (h) administering a therapeutically effective portion of the third population of TILs to the subject or patient with the cancer; (i) optionally, at any time prior to the administering step (h), genetically modifying the TIL population so that the administered third TIL population comprises genetically modified TILs that comprise a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

7. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TILs), said method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer in the patient or subject; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) performing a first priming expansion by culturing the TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of time of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (d) optionally restimulating the second TIL population with OKT-3; (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers; (f) performing a second rapid expansion of the modified second TIL population by culturing in a second culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of time of up to about 14 days to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising the genetic modification that reduces or increases expression of the one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers; (g) harvesting the third population of TILs; (h) administering a therapeutically effective portion of the third population of TILs to the subject or patient with the cancer; (i) optionally, at any time prior to the administering step (h), genetically modifying the TIL population so that the administered third TIL population comprises genetically modified TILs that comprise a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

8. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) performing a first priming expansion of the TIL population by culturing the TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, and producing the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (d) performing a second rapid expansion of the second TIL population by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (b), and the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the therapeutic TIL population, wherein the third TIL population is the therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area; (e) harvesting the therapeutic TIL population obtained from step (d); (f) transferring the harvested TIL population from step (e) to an infusion bag; (g) optionally, genetically modifying the TIL population at any time prior to the harvesting step (e), such that the therapeutic TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

9. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject or patient by processing a tumor sample obtained from the tumor into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) optionally adding said TIL population to a closed system; (d) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is optionally performed in a sealed container that provides a second gas permeable surface area, and wherein the transition from step (d) to step (e) optionally occurs without opening the system; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the system; (h) optionally, genetically modifying the TIL population at any time prior to the harvesting step (f) such that the third TIL population comprises a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

10. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: (a) obtaining a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; b) selecting from said first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) optionally adding said TIL population to a closed system; (d) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) optionally occurs without opening the system to produce the third TIL population; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the system; (h) optionally, at any time prior to the harvesting step (f), genetically modifying the TIL population so that the third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers.

11. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) optionally adding said TIL population to a closed system; (d) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) optionally occurs without opening the system to produce the third TIL population; (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (e) to (f) optionally occurs without opening the system; (h) optionally, at any time prior to the harvesting step (f), genetically modifying the TIL population so that the third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

12. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: (a) resecting a tumor from a cancer in a subject or patient, said tumor optionally comprising a first TIL population from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from said cancer; (b) processing the tumor into a plurality of tumor fragments or tumor digests; (c) enzymatically digesting the plurality of tumor fragments to obtain the first population of TILs; (d) selecting from the first TIL population of (c) a TIL population that expresses at least one checkpoint inhibitor, including CD39 and CD103; (e) optionally adding said TIL population to a closed system; (f) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and wherein the transition from step (e) to step (f) optionally occurs without opening the system; (g) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (f) to step (g) optionally occurs without opening the system to produce the third TIL population; (h) harvesting the third population of TILs obtained from step (g), wherein the transition from step (g) to step (h) optionally occurs without opening the system; (i) transferring the harvested third population of TILs from step (h) to an infusion bag, wherein the transition from step (h) to (i) optionally occurs without opening the system; (j) optionally, at any time prior to the harvesting step (h), genetically modifying the TIL population so that the third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

13. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a subject or patient; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) contacting the TIL population with a first cell culture medium; (d) performing a first expansion of the TIL population in the first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, the first cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population 7-8 days after initiation of the rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (an anti-CD3 antibody), and APC, the rapid expansion being performed over a period of 14 days or less, and optionally, the rapid second expansion being allowed to proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after initiation of the rapid second expansion; (f) harvesting the third population of TILs; (g) optionally, at any time prior to the harvesting step (f), genetically modifying the TIL population so that the third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

14. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, said method comprising: a) resecting a tumor from a cancer in a subject or patient, said tumor optionally comprising a first TIL population from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from said cancer; (b) fragmenting the tumor into tumor fragments or tumor digests; (c) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) contacting the tumor fragment with a first cell culture medium; (d) performing an initial expansion (or first expansion by priming) of the TIL population in the first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally antigen-presenting cells (APCs), and the first expansion by priming occurs over a period of 1 to 8 days; (e) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), 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 initiation of the rapid second expansion; (f) harvesting the third population of TILs; (g) optionally, at any time prior to said harvesting (f), genetically modifying the TIL population so that the harvested third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

15. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) performing a first priming expansion of the TIL population by culturing the TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, and producing the second TIL population, wherein the second TIL population is more numerous than the first TIL population; (d) performing a second rapid expansion of the second TIL population by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (e) harvesting the therapeutic TIL population obtained from step (c); (g) optionally, genetically modifying the TIL population at any time prior to the harvesting step (e), such that the third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

16. 16. The method of claim 15, wherein in step (c), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (d) is greater than the number of APCs in the culture medium in step (c).

17. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from a cancer in a patient or subject; (b) selecting from the first TIL population of (a) a TIL population that expresses at least CD39 and CD103; (c) performing a first priming expansion of the TIL population by culturing the TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of time of about 1 to 11 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (d) optionally restimulating said second TIL population with OKT-3; and (e) genetically modifying the second TIL population to produce a modified second TIL population, wherein the modified second TIL population comprises a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers; (f) performing a second rapid expansion of the modified second TIL population by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of time of not more than about 14 days to obtain a therapeutic TIL population, wherein the third TIL population is a therapeutic TIL population comprising the genetic modification that reduces or increases expression of the one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers; (g) harvesting a third population of TILs.

18. 18. The method of any one of claims 1 to 17, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

19. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first expansion by priming by culturing a first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, wherein said TIL population expresses at least CD39 and CD103; optionally, the TILs include antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 1 to 11 days to obtain the second TIL population, and the second TIL population is more numerous than the first TIL population; (b) performing a second rapid expansion of the second TIL population by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (c) harvesting the third population of TILs obtained from step (b); and (d) genetically modifying the TIL population, the second TIL population, and / or the third TIL population at any time prior to the harvesting step (c) so that the harvested third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with said T cell exhaustion markers.

20. 20. The method of claim 19, wherein in step (a), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

21. 1. A method for expanding T cells, comprising: (a) performing a first expansion by priming of a first TIL population obtained from a donor by culturing the first TIL population to result in growth and to prime activation of a first T cell population, wherein the first TIL population is a TIL population, and the TIL population expresses at least CD39 and CD103; (b) after activation of the first TIL population primed in step (a) begins to decay, performing a rapid second expansion of the first TIL population by culturing the first TIL population to result in growth and promote activation of the first T cell population to obtain a second T cell population; (c) harvesting the second population of T cells; (d) genetically modifying the first TIL population and / or the second TIL population such that the harvested second TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers.

22. 1. A method for expanding T cells, comprising: (a) a first expansion by priming a first T cell population from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor in a donor by culturing the first T cell population to result in growth and prime activation of the first T cell population, wherein the first T cell population is a T cell population that expresses at least CD39 and CD103; (b) after the activation of the first T cell population primed in step (a) begins to decay, performing a rapid second expansion of the first T cell population by culturing the first T cell population to result in growth and promote the activation of the first T cell population to obtain a second T cell population; (c) harvesting the second population of T cells; (d) genetically modifying the first population of T cells and / or the second population of TILs such that the harvested second population of T cells comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers.

23. 23. The method of any one of claims 1 to 22, wherein the one or more T cell exhaustion markers are selected from PD-1 and CD39, and combinations thereof.

24. The method of any one of claims 1 to 23, wherein the one or more factors phenotypically associated with T cell exhaustion markers is CD103.

25. 25. The method of any one of claims 1 to 24, further comprising an additional step comprising selecting from the TIL population a TIL population that expresses at least one protein selected from the group consisting of OX40, 4-1BB, and combinations thereof.

26. 26. The method of claim 25, wherein the additional selecting step occurs before the first dilation.

27. 26. The method of claim 25, wherein the additional selecting step occurs before the first expansion by priming.

28. 26. The method of claim 25, wherein the additional selecting step occurs during the first expansion by priming.

29. 29. The method of any one of claims 25 to 28, wherein the additional selecting step occurs during days 0, 1, 2, 3, 4, and / or 5, or during the first expansion by priming, where OKT-3 has not yet been added.

30. 20. The method of any one of claims 1-15, 17, or 19, further comprising a second selection of a TIL population from said second TIL population that expresses at least one protein selected from the group consisting of OX40, 4-1BB, and combinations thereof.

31. 17. The method of any one of claims 1-4, 9-13, or 16, wherein the modifying is performed on the second TIL population from the first expansion, or the third TIL population from the second expansion, or both.

32. 21. The method of any one of claims 5, 6, 8, 14-16, or 18-20, wherein the modifying is performed on the second TIL population from the priming first expansion, or the third TIL population from the rapid second expansion, or both.

33. 17. The method of any one of claims 1-4, 9-13, or 16, wherein the modifying is performed on the second TIL population from the first expansion and on the second TIL population before the second expansion.

34. 21. The method of any one of claims 5, 6, 8, 14-16, or 18-20, wherein the modifying is performed on the second TIL population from the priming first expansion, the second TIL population before the rapid second expansion, or both.

35. 17. The method of any one of claims 1-4, 9-13, or 16, wherein said modifying is performed on the third population of TILs from the second expansion.

36. 21. The method of any one of claims 5, 6, 8, 14-16, or 18-20, wherein said modifying is performed on the third TIL population from the rapid second expansion.

37. 23. The method of any one of claims 1-6, 8-16, or 18-22, wherein said modifying is performed after said harvesting.

38. 17. The method of any one of claims 1 to 4, 9 to 13, or 16, wherein the first expansion is performed for a period of about 11 days.

39. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the first expansion by priming is carried out over a period of about 11 days.

40. 17. The method of any one of claims 1-4, 9-13, or 16, wherein the IL-2 is present in the cell culture medium in the first expansion at an initial concentration of 1000 IU / mL to 6000 IU / mL.

41. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the IL-2 is present in the cell culture medium at an initial concentration of 1000 IU / mL to 6000 IU / mL in the first expansion by priming.

42. 17. The method of any one of claims 1-4, 9-13, or 16, wherein in the second expansion step, the IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

43. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein in the rapid second expansion step, the IL-2 is present at an initial concentration of 1000 IU / mL to 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

44. 17. The method of any one of claims 1 to 4, 9 to 13, or 16, wherein the first expansion is performed using a gas-permeable container.

45. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the first expansion by priming is performed using a gas-permeable container.

46. 17. The method of any one of claims 1 to 4, 9 to 13, or 16, wherein the second expansion is performed using a gas-permeable container.

47. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the rapid second expansion is performed using a gas-permeable container.

48. 17. The method of any one of claims 1-4, 9-13, or 16, wherein the cell culture medium of the first expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

49. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the cell culture medium of the first expansion by priming further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

50. 17. The method of any one of claims 1-4, 9-13, or 16, wherein the cell culture medium of the second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

51. 23. The method of any one of claims 5-8 or 14-22, wherein the cell culture medium of the rapid second expansion further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

52. 8. The method of any one of claims 1-7, further comprising treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.

53. The non-myeloablative lymphodepletion regimen comprises cyclophosphamide at 60 mg / m ２ / day for 2 days, followed by fludarabine at 25 mg / m ２ 53. The method of claim 52, comprising administering at a dose of 0.1 mg / day for three days.

54. The non-myeloablative lymphodepletion regimen comprises cyclophosphamide at 60 mg / m ２ / day and fludarabine at a dose of 25 mg / m ２ / day for 2 days, followed by fludarabine at 25 mg / m ２ 53. The method of claim 52, comprising administering at a dose of 0.1 mg / day for three days.

55. The non-myeloablative lymphodepletion regimen comprises cyclophosphamide at 60 mg / m ２ / day and fludarabine at a dose of 25 mg / m ２ / day for 2 days, followed by fludarabine at 25 mg / m ２ 53. The method of claim 52, comprising administering at a dose of 0.1 mg / day for one day.

56. 56. The method of any one of claims 53 to 55, wherein the cyclophosphamide is administered with mesna.

57. 57. The method of any one of claims 1-7 or 52-56, further comprising treating said patient with an IL-2 regimen starting the day after administration of said TILs to said patient.

58. 57. The method of any one of claims 1-7 or 52-56, further comprising treating said patient with an IL-2 regimen starting on the same day as administration of said TILs to said patient.

59. 59. The method of claim 57 or 58, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

60. A therapeutically effective TIL population was administered, approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ 60. The method of any one of claims 1 to 7 or 52 to 59, comprising a TIL.

61. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the priming first expansion and the rapid second expansion are carried out over a period of 21 days or less.

62. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the priming first expansion and rapid second expansion are carried out over a period of not more than 16 or 17 days.

63. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the first expansion by priming is carried out over a period of not more than 7 or 8 days.

64. 23. The method of any one of claims 5 to 8 or 14 to 22, wherein the rapid second expansion is performed over a period of 11 days or less.

65. 17. The method of any one of claims 1 to 4, 9 to 13, or 16, wherein the first expansion and the second expansion are each performed separately within a period of 11 days.

66. 18. The method of claim 7 or 17, wherein steps (a) through (f) are carried out within about 26 days.

67. The genetically modified TILs may express any of the following proteins: CTLA-4, Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, S 67. The method of any one of claims 1-66, further comprising an additional genetic modification that reduces or increases expression of one or more of the immune checkpoint genes selected from the group comprising: MAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, and TOX.

68. 68. The method of claim 67, wherein the one or more immune checkpoint genes are selected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.

69. 69. The method of any of claims 1-68, wherein the genetically modified TILs further comprise an additional genetic modification that enhances expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population, wherein the immune checkpoint gene(s) is / are selected from the group comprising CCR2, CCR4, CXCR2, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or NOTCH ligand mDLL1.

70. 70. The method of any of claims 1-69, wherein said genetic modifying step is carried out using a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks in said one or more immune checkpoint genes.

71. The method of any one of claims 1 to 70, wherein the genetic modification is carried out using one or more methods selected from the CRISPR method, the TALE method, the zinc finger method, and combinations thereof.

72. 72. The method of claim 71, wherein the method comprises a CRISPR method.

73. 73. The method of claim 72, wherein the CRISPR method is a CRISPR / Cas9 method.

74. 72. The method of claim 71, wherein said genetic modifying comprises a TALE method.

75. 72. The method of claim 71, wherein said genetic modification comprises zinc finger technology.

76. 76. The method of any of claims 1-75, wherein processing the tumor sample obtained from the subject into a tumor digest comprises incubating the tumor sample in an enzyme medium.

77. 76. The method of any one of claims 1 to 75, wherein processing the tumor sample obtained from the subject into a tumor digest further comprises mechanically disrupting the tumor sample to dissociate the tumor sample.

78. 76. The method of any of claims 1-75, wherein processing a tumor sample obtained from the subject into a tumor digest further comprises purifying the dissociated tumor sample using density gradient separation.

79. 77. The method of claim 76, wherein the enzyme medium comprises DNase.

80. 80. The method of claim 76 or 79, wherein the enzyme medium contains 30 units / mL of DNase.

81. 81. The method of any one of claims 76 or 79-80, wherein the enzyme medium comprises collagenase.

82. 82. The method of any one of claims 76 or 79-81, wherein the enzyme medium comprises 1.0 mg / mL collagenase.

83. 83. The method of any of claims 1-82, wherein the harvested therapeutic TIL population comprises sufficient TILs for use in administering a therapeutically effective dose to a subject.

84. The therapeutically effective dose is about 1×10 ９ ~Approx. 9×10 １０ 84. The method of any preceding claim, comprising TIL.

85. The therapeutically effective population of TILs is administered and is about 2.3 x 10 １０ ~Approx. 13.7×10 １０ 84. The method of any preceding claim, comprising TIL.

86. The method of any one of claims 1 to 85, wherein the APC is a peripheral blood mononuclear cell (PBMC).

87. 87. The method of any preceding claim, wherein the therapeutic TIL population harvested in step (e) exhibits an increased subpopulation of CD8+ cells compared to the first and / or second TIL populations.

88. 88. The method of any preceding claim, wherein the PBMCs are replenished at a ratio of about 1:25 TILs:PBMCs.

89. 89. The method of any one of claims 1 to 88, wherein the first expansion and the second expansion are performed separately within a period of 11 to 12 days each.

90. 90. The method of any of claims 1-89, wherein steps (a) through (e), (f), or (g) are carried out for about 10 days to about 24 days.

91. 91. The method of any of claims 1-90, wherein steps (a)-(e), (f), or (g) are carried out for about 15 days to about 24 days.

92. 92. The method of any of claims 1-91, wherein steps (a)-(e), (f), or (g) are carried out in about 20 days to about 24 days.

93. 93. The method of any of claims 1-92, wherein steps (a)-(e), (f), or (g) are carried out for about 20 days to about 22 days.

94. 94. The method of any preceding claim, wherein the second TIL population is at least 50 times more numerous than the first TIL population.

95. 1. A method of treating cancer in a patient or subject in need thereof, comprising administering a population of tumor infiltrating lymphocytes (TILs), said method comprising: (a) selecting, from said first TIL population obtained and / or received from surgical resection, needle biopsy, core biopsy, mini-biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from said cancer in said patient or said subject, a TIL population that expresses at least one protein selected from the group consisting of CD39, CD103, and combinations thereof; (b) further selecting from the population of TILs a population of TILs that expresses at least one protein selected from the group consisting of OX40, 4-1BB, and combinations thereof; (c) optionally adding said TIL population to a closed system; (d) performing a first expansion of the TIL population by culturing it in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is optionally performed in a sealed container that provides a first gas permeable surface area, and wherein the first expansion is performed for about 3 to 11 days to obtain the second TIL population, and wherein the transition from step (c) to step (d) optionally occurs without opening the system; (e) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 11 days to obtain the third TIL population, and the second expansion is optionally performed in a sealed container that provides a second gas permeable surface area, and the transition from step (d) to step (e) optionally occurs without opening the system to produce the third TIL population.

96. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a cancer in a subject by processing a tumor sample obtained from the tumor into a plurality of tumor fragments or by processing a tumor sample obtained from the subject into a tumor digest; (b) optionally, in a closed system, selecting from said first TIL population of (a) a TIL population that expresses at least one protein selected from the group consisting of CD39, CD103, and combinations thereof; (c) further selecting from the TIL population a TIL population that expresses at least one protein selected from the group consisting of OX40, 4-1BB, and combinations thereof; (d) performing a first priming expansion by culturing the TIL population in a first cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, and producing the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (e) performing a second rapid expansion of the second TIL population by culturing the second TIL population in a second culture medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (b), and the rapid second expansion is performed for a second period of about 1 to 11 days to obtain a therapeutic TIL population, wherein the third TIL population is the therapeutic TIL population, and the rapid second expansion is performed in a container comprising a second gas permeable surface area.

97. (f) harvesting the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) optionally occurs without opening the system; and (g) transferring the harvested third population of TILs from step (f) to an infusion bag, wherein the transition from step (e) to (f) optionally occurs without opening the system; (h) cryopreserving the infusion bag containing the harvested TIL population from step (g) using a cryopreservation process; (i) administering a therapeutically effective dose of the third population of TILs to the subject from the infusion bag of step (h); 97. The method of claim 95 or 96, further comprising: (j) optionally genetically modifying the TIL population any time prior to the administering step (i), such that the administered third TIL population comprises genetically modified TILs comprising a genetic modification that reduces or increases expression of one or more T cell exhaustion markers and / or one or more factors phenotypically associated with the T cell exhaustion markers.

98. The method of any one of claims 95 to 97, further comprising the method of any one of claims 1 to 94.

99. The TIL population according to any of the methods of claims 1 to 98.

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