Expansion of TILS from cryopreserved tumor samples

Abstract

Methods of expanding tumor infiltrating lymphocytes (TILs) from cryopreserved tumor tissue and methods of using the expanded TILs in the treatment of human diseases, including cancers, are disclosed. Preparing the tumor tissue for freezing includes fragmenting the tumor tissue, and incubating the fragments in a cryopreservation medium. In an embodiment, the fragments may be incubated in the cryo preservation medium at 2°C to 8°C for about 30 to about 80 minutes. Freezing the fragments may be done by flash freezing using the vapor phase of liquid nitrogen, e.g. using a dry cryoshipper. In some embodiments, compositions of cryopreserved tumor tissues are disclosed.

Description

DEMANDE OU BREVET VOLUMINEUX LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND PLUS D'UN TOME. CECI EST LE TOME 1 DE 2 CONTENANT LES PAGES 1 À 183 NOTE: Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS / PATENTS THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE THAN ONE VOLUME THIS IS VOLUME 1 OF 2 CONTAINING PAGES 1 TO 183 NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME : NOTE POUR LE TOME / VOLUME NOTE: EXPANSION OF TILS FROM CRYOPRESERVED TUMOR SAMPLES CROSS REFERENCE TO RELATED APPLICATIONS

[001] This international PCT application claims priority to U.S. Provisional Application No. 62 / 733,937, filed on September 20, 2018, and U.S. Provisional Application No. 62 / 879,881, filed on July 29, 2019. FIELD OF THE INVENTION

[002] The invention described herein relates generally to the expansion of lymphocytes and more particularly, but not exclusively, to expansion of lymphocytes from cryopreserved tumor tissue. BACKGROUND OF THE INVENTION

[003] Treatment of refractory cancers using adoptive transfer of tumor infiltrating lymphocytes (TILs) represents a potentially powerful approach to treat patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires a large number of TILs; therefore, a robust and reliable process is needed for manufacturing and commercialization. This scaling for commercialization has been a profound challenge because of the legion technical, logistical, and regulatory issues with cell expansion. IL-2-based TIL expansion followed by a "rapid expansion process" (REP) has become a preferred method for TIL expansion because of its 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. A REP can result in a 1,000-fold expansion of TILs over a 14-day period, although it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs that have undergone a REP procedure have produced successful adoptive cell therapy following host immunosuppression in some melanoma patients.

[004] Current TIL manufacturing processes are limited by duration, cost, sterility concerns, and other factors described herein such that the potential to commercialize such processes is severely limited. Among the many limitations of the current processes is the dependence on fresh tumor tissue to initiate manufacturing, which often necessitates expensive and costly local manufacturing facilities because of the difficulty in shipping fresh tumor longer distances. There is an urgent need to provide TIL manufacturing processes and therapies that are not dependent on fresh tumor tissue and that are appropriate for commercial scale manufacturing and regulatory approval for use in geographically widely dispersed human patients. SUMMARY OF THE INVENTION

[005] In an embodiment, the invention provides methods of TIL expansion that utilize frozen tumor tissue as the starting TIL source material to produce therapeutic populations of TILs.

[006] In an embodiment, the invention provides a method for cryopreserving tumor tissue for the manufacture of tumor infiltrating lymphocytes (TILs) comprising: (i) fragmenting the tumor tissue; (ii) incubating the fragments in a cryopreservation medium; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

[007] In an embodiment, the invention provides a cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by a process comprising the steps of: (i) fragmenting a tumor sample; (ii) incubating the fragments in a cryopreservation medium; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

[008] In an embodiment, the invention provides a cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by a process comprising the steps of: (i) fragmenting a tumor sample; (ii) incubating the fragments for about 20 minutes to about 70 minutes at a temperature in the range of about 2°C to about 8°C in a cryopreservation medium comprising about 10% v / v dimethylsulfoxide; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

[009] In an embodiment, the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising incubating the tumor tissue for at least 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing; (b) thawing the tumor tissue; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (d) removing at least a plurality of the TILs; and (e) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[0010] In an embodiment, the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) obtaining tumor tissue from a patient; (b) incubating the tumor tissue for at least 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C; (c) freezing the tumor tissue; (d) storing the tumor tissue in a frozen state; (b) thawing the tumor tissue; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (d) removing at least a plurality of the TILs; and (e) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[0011] In some embodiments, the storage medium comprises 2 to 12 % v / v (volume:volume) dimethylsulfoxide (DMSO). In some embodiments the storage medium comprises 5% v / v DMSO. In some embodiments the storage medium comprises 10% v / v DMSO. In some embodiments, the storage medium comprises between 5 and 10 % v / v DMSO. In some embodiments the storage medium comprises a percentage of DMSO selected from the group consisting of 1% v / v, 2% v / v, 3% v / v, 4% v / v, 5% v / v, 6% v / v, 7% v / v, 8% v / v, 9% v / v, 10% v / v, 11% v / v, 12% v / v, 13% v / v, 14% v / v, and 15% v / v. In some of the foregoing embodiments, the remainder of the storage medium is an aqueous medium.

[0012] In some embodiments, the storage medium comprises 2 to 12 % w / w (weight:weight) dimethylsulfoxide (DMSO). In some embodiments the storage medium comprises 5% w / w DMSO. In some embodiments the storage medium comprises 10% w / w DMSO. In some embodiments, the storage medium comprises between 5 and 10 % w / w DMSO. In some embodiments the storage medium comprises a percentage of DMSO selected from the group consisting of 1% w / w, 2% w / w, 3% w / w , 4% w / w , 5% w / w , 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11% w / w, 12% w / w, 13% w / w, 14% w / w, and 15% w / w. In some of the foregoing embodiments, the remainder of the storage medium is an aqueous medium.

[0013] In some embodiments, the tumor tissue is incubated for at least 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for between 30 minutes and about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 45 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing.

[0014] In some embodiments, the storage medium comprises about 5% v / v DMSO and the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the storage medium comprises about 10% v / v DMSO and the tumor tissue is incubated for at least 30 minutes to about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing.

[0015] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (1) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (iv) freezing the vessel; and (v) storing the vessel such that the vessel remains frozen; (b) thawing the vessel; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (d) removing at least a plurality of the TILs; (e) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[0016] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient; (b) trimming the tumor tissue to remove excess non-tumor tissue; (b) placing the tumor tissue in a closable vessel containing a storage medium; (c) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (d) freezing the vessel; (e) storing the vessel such that the vessel remains frozen; (d) thawing the vessel; (f) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (g) removing at least a plurality of the TILs; and (h) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[0017] In some embodiments, the fresh tumor tissue is trimmed to between 1.5 mm and 6 mm in diameter and between 1.5 mm and 6 mm in thickness. In yet other embodiments, the fresh tumor tissue is trimmed to about 6 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6 mm.

[0018] In some embodiments, the storage medium further comprises an antibacterial agent. In some embodiments the antibacterial agent is gentamicin. In some embodiments the storage medium comprises gentamicin at a concentration of at least 50 µg / mL. In some embodiments the storage medium comprises an antibacterial agent, an antifungal agent, and combinations thereof. In some embodiments, the antifungal agent is amphotericin B and is present from about 0.25 µg / mL to about 2.5 µg / mL. In some embodiments, the antifungal agent is fungin and is present from about 5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>g / mL to about 50 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>g / mL.

[0019] In some embodiments, the fresh tumor tissue is first washed in Hank's Balanced Salt Solution (HBSS) or another suitable medium or solution before performing the freezing step of the method. In some embodiments, the washing comprises at least three serial washes of at least three minutes each, wherein the HBSS is replaced after each wash.

[0020] In some embodiments the thawing step comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[0021] In some embodiments, the freezing step comprises freezing the vessel at a temperature of about -125 °C to about -195 °C. In some embodiments, the vessel is frozen at a temperature of about -125 °C to about -150 °C; in some embodiments the vessel is frozen at a temperature of about -125 °C to about -145 °C; in yet further embodiments, the vessel is frozen at a temperature of about -135 °C.

[0022] In some embodiments, the method is performed with a fresh tumor sample from a human subject.

[0023] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC), and HPV-positive tumor tissue.

[0024] In some embodiments, the method further comprises a split between the first expansion step (c) and the second expansion step (e). In an embodiment, the split occurs on day 16. In some embodiments, the first culture step is complete in about 11 days. In other embodiments, the first expansion (or first culture) step is complete in about 7 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 7 days and the second expansion (or second culture) step is complete in about 14 days. In some embodiments, steps (c) through (e) are complete in about 22 days. In yet other embodiments, steps (c) through (e) are complete in 21 days; in some embodiments steps (c) through (e) are complete in about 20 days; and in some embodiments steps (c) through (e) are complete in about 16 days.

[0025] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof are provided, such methods comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (iv)freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (b) thawing the tumor tissue; (c) adding the tumor fragments into a closed system; (d) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein 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 population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the therapeutic population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested TIL population from step (g) using a cryopreservation process; and (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject.

[0026] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof are provided, such methods comprising administering expanded tumor infiltrating lymphocytes (TLLs) comprising: (a) obtaining tumor tissue from the subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) freezing the vessel using the vapor phase of liquid nitrogen; (f) storing the vessel at the vapor phase of liquid nitrogen temperature; (g) thawing the tumor tissue; (h) adding the tumor tissue into a closed system; (i) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (i) to step (j) occurs without opening the system; (k) harvesting the therapeutic population of TILs obtained from step (j), wherein the transition from step (j) to step (k) occurs without opening the system; (1) transferring the therapeutic population of TILs harvested in step (k) to an infusion bag, wherein the transfer from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (1) using a cryopreservation process; and (n) administering a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (m) to the subject.

[0027] In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC) glioblastoma, endometrial cancer, thyroid cancer, colorectal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, melanoma, refractory melanoma, metastatic melanoma, and non-small cell lung carcinoma. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment.

[0028] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising incubating the tumor tissue for about 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing; (c) thawing the tumor tissue; (d) treating the tumor tissue in a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 antibody to provide TILs; (e) removing at least a plurality of the TILs; and (f) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[0029] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) incubating the tumor tissue for about 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C; (c) freezing the tumor tissue; (d) storing the tumor tissue in a frozen state: (e) thawing the tumor tissue; (f) treating the tumor tissue in a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 antibody to provide TILs; (g) removing at least a plurality of the TILs; and (h) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[0030] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 30 minutes; (iv) freezing the vessel; and (v) storing the vessel such that the vessel remains frozen; (c) thawing the vessel; (d) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (e) removing at least a plurality of the TILs; and (f) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[0031] In some embodiments, the fresh tumor tissue is first washed in Hank's Balanced Salt Solution (HBSS) before performing the freezing step of the method. In some embodiments, the washing comprises at least three serial washes of at least three minutes each, wherein the HBSS is replaced after each wash.

[0032] In some embodiments the thawing step comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[0033] In some embodiments, the freezing step comprises freezing the vessel at a temperature of about -125°C to about -195°C. In some embodiments, the vessel is frozen at a temperature of about -125°C to about -150°C; in some embodiments the vessel is frozen at a temperature of about -125°C to about -145°C; in yet further embodiments, the vessel is frozen at a temperature of about -135°C.

[0034] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC), and HPV-positive tumor tissue.

[0035] In some embodiments, the method further comprises a split between the first expansion (or first culture) step and the second expansion (or second culture) step. In an embodiment, the split occurs on day 16. In some embodiments, the first culture step is complete in about 11 days. In other embodiments, the first expansion (or first culture) step is complete in about 7 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 7 days and the second expansion (or second culture) step is complete in about 14 days. In some embodiments, steps (d) through (f) or steps (i) through (k), as applicable, are complete in about 22 days. In yet other embodiments, steps (d) through (f) or steps (i) through (k), as applicable, are complete in about 21 days; in some embodiments steps (d) through (f) or steps (i) through (k), as applicable, are complete in about 20 days; and in some embodiments steps (d) through (f) or steps (i) through (k), as applicable, are complete in about 16 days.

[0036] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof are provided, such methods comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising: (a) obtaining tumor tissue from the subject, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii)incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 30 minutes; (iv)freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (c) thawing the tumor tissue; (d) adding the tumor tissue into a closed system; (e) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (e) to step (f) occurs without opening the system; (g) harvesting the therapeutic population of TILs obtained from step (f), wherein the transition from step (f) to step (g) occurs without opening the system; (h) transferring the therapeutic population of TILs harvested from step (g) to an infusion bag, wherein the transfer from step (g) to (h) occurs without opening the system; (i) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (h) using a cryopreservation process; and (i) administering a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (i) to the subject.

[0037] In some embodiments, the fresh tumor tissue is first washed in Hank's Balanced Salt Solution (HBSS) before performing the freezing step of the method. In some embodiments, the washing comprises at least three serial washes of at least three minutes each, wherein the HBSS is replaced after each wash.

[0038] In some embodiments the thawing step comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[0039] In some embodiments, the freezing step comprises freezing the vessel at a temperature of about -125°C to about -195°C. In some embodiments, the vessel is frozen at a temperature of about -125°C to about -150°C; in some embodiments the vessel is frozen at a temperature of about -125°C to about -145°C; in yet further embodiments, the vessel is frozen at a temperature of about -135°C.

[0040] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC), and HPV-positive tumor tissue.

[0041] In some embodiments, the method further comprises a split between the first expansion step and the second expansion step. In an embodiment, the split occurs on day 16. In some embodiments, the first expansion step is complete in about 11 days. In other embodiments, the first expansion step is complete in about 7 days. In yet other embodiments, the first expansion step is complete in about 7 days and the second expansion step is complete in about 14 days. In some embodiments, steps (e) through (g) are complete in about 22 days. In yet other embodiments, steps (e) through (g) are complete in about 21 days; in some embodiments steps (e) through (g) are complete in about 20 days; and in some embodiments steps (e) through (g) are complete in about 16 days.

[0042] In some embodiments, an expansion method according to the present invention is completed in about 16 days. In some embodiments, an expansion method comprises a first expansion step (a preREP step) and a second expansion step (a REP step). In an embodiment, the first expansion step is from about 3 to about 5 days. In another embodiment, the first expansion step is about 3 days. In another embodiment, the first expansion step is about 5 days. In some embodiments, the second expansion step is from about 11 to about 13 days. In another embodiment, the second expansion step is about 11 days. In another embodiment, the second expansion step is about 13 days. In some embodiments, the first expansion step is about 5 days and the second expansion step is about 11 days. In some embodiments, the first expansion step is about 3 days and the second expansion step is about 13 days.

[0043] In some embodiments, the TILs are split into additional containers during the second expansion step. In some embodiments, the TILs are split on day 5 of the second expansion step. In some embodiments, the TILs are split on day 6 of the second expansion step. In some embodiments, the second expansion step is about 11 days and the TILs are split on day 5 of the second expansion step. In some embodiments, the second expansion step is about 13 days and the TILs are split on day 6 of the second expansion step.

[0044] In some embodiments, the first expansion step is 3 days, the second expansion step is 13 days, and the TILs are split on day 6 of the second expansion step (day 9 of the total process). In some embodiments, the first expansion step is 5 days, the second expansion step is 11 days, and the TILs are split on day 5 of the second expansion step (day 10 of the total process).

[0045] In some embodiments, feeder cells are used during the second expansion step. In an embodiment, in a <semantics>1 / 100<annotation encoding="application / x-tex">1 / 100< / annotation>< / semantics>th scale process, about 2-50 x <semantics>106<annotation encoding="application / x-tex">10^6< / annotation>< / semantics> feeder cells are used during the second expansion step. In another embodiment, about <semantics>25×106<annotation encoding="application / x-tex">25 \times 10^6< / annotation>< / semantics> feeder cells are used. In another embodiment, about 50 x 106 feeder cells are used. In some embodiments, in a full scale process, about 2-50 <semantics>×109<annotation encoding="application / x-tex">\times 10^9< / annotation>< / semantics> feeder cells are used. In some embodiments, in a full scale process, about <semantics>2.5×109<annotation encoding="application / x-tex">2.5 \times 10^9< / annotation>< / semantics> or about <semantics>5×109<annotation encoding="application / x-tex">5 \times 10^9< / annotation>< / semantics> feeder cells are used. In some embodiments, the feeder cells are PBMCs.

[0046] In some embodiments, the second expansion step is seeded with from about <semantics>5×106<annotation encoding="application / x-tex">5 \times 10^6< / annotation>< / semantics> to about 200 x 106 TIL. In some embodiments, the second expansion step is seeded with about 5% to about 25% of the TILs from the first expansion step. In some embodiments, the second expansion step is seeded with about 10% to about 20% of the TILs from the first expansion step. In some embodiments, the second expansion step is seeded with about 10% of the TILs from the first expansion step.

[0047] Other embodiments include combinations and variations of these compositions and methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

[0049] Figure 1: Depicts the major steps of an embodiment of the 2A process, including the optional cryopreservation of fresh tumor tissue after fragmentation in Step B. The total time from the initiation of the first expansion culture to the end of Step E is about 22 days.

[0050] Figure 2: Panels A through C depict the various steps of an embodiment of the TIL manufacturing process, including cryopreservation of fresh tumor fragments allowing later initiation of preREP culture.

[0051] Figure 3: Depicts an embodiment of TIL manufacturing, including the cryopreservation of fresh tumor fragments that are then later used to initiate a preREP culture.

[0052] Figure 4: Depicts an embodiment of TIL manufacturing, including the cryopreservation of fresh tumor fragments that are then later used to initiate a pre-REP culture.

[0053] Figure 5: Generally compares an embodiment of Process 1C to an exemplary embodiment of Process 2A. Process 2A contemplates initiation of pre-REP cultures from either fresh tumor tissue fragments or thawed cryopreserved tumor tissue fragments.

[0054] Figure 6: Further compares embodiments of Process 1C to embodiments of Process 2A.

[0055] Figure 7: Depicts an embodiment of the 2A process wherein the REP is initiated "early."

[0056] Figure 8: Compares the total viable cells derived from a Gen 2A process and a modified Gen 2A process, noted as "Gen 2.2" wherein the pre-REP cultures are initiated with fresh, flash frozen, or frozen following 30 minute or 60 minute incubations in storage medium at 2°C to 8°C. Cell counts are at the end of the pre-REP culture.

[0057] Figure 9: Compares the total viable cells derived from a Gen 2A process and a modified Gen 2A process, noted as "Gen 2.2" wherein the pre-REP cultures are initiated with fresh, flash frozen, or frozen following 30 minute or 60 minute incubations in storage medium at 2°C to 8°C. Cell counts at the end of the REP culture.

[0058] Figure 10: Compares IFN-γ produced by cells derived from a Gen 2A process and a modified Gen 2A process, noted as "Gen 2.2" wherein the pre-REP cultures are initiated with fresh, flash frozen, or frozen following 30 minute or 60 minute incubations in storage medium at 2°C to 8°C.

[0059] Figure 11: Depicts the major steps of a Gen 2 embodiment compared to the major steps of a Gen 2.2 embodiment.

[0060] Figure 12: Depicts the major steps of the 1 / 100th scale TIL production process.

[0061] Figure 13: Figures 13A-13E depict total viable cells (TVC) for tumor samples processed from 5 different patients and collected during pre-REP. TVC was measured using fresh (Gen2 control) and frozen tumor samples. TVC was recorded on day 11 for fresh tumor samples or day 7 for frozen tumor samples. Figure 13A (patient M1125) shows the only patient whose tumor samples were subjected to different freezing methods: flash freezing in the vapor phase of liquid nitrogen, and incubation at 2-8° Celsius for either 30 or 60 minutes prior to flash freezing in the vapor phase of liquid nitrogen. For the remaining patients S13018, OV8022, T6042, and O8026 (FIGS. 13B-13E, respectively), the tumor samples were incubated at 2-8° for 60 minutes prior to flash freezing. For each patient, tumor samples were sectioned in 3mm diameter fragments and 4-6 fragments were used for culture. Each bar on the graph represents TVC extrapolated to full scale (multiplied by 10). In FIG 13A, frozen, early pre-REP culture yielded about 1% TVC as compared to fresh, FIG. 13B, about 25% TVC as compared to fresh, FIG. 13C, about 200% as compared to fresh, FIG 13D, about 96% as compared to fresh, and FIG. 13E, about 3% as compared with fresh. The data indicate that TVC from frozen tumor samples on Day 7 is comparable to TVC from fresh tumor samples on Day 11.

[0062] Figure 14: Figures 14A-14E depict TVC for tumor samples from the same 5 patients as Figure 13, collected during early REP. Cells were harvested on Day 11 (fresh) and Day 7 (frozen) as described in Figure 13. In FIG 14A, frozen, early REP culture yielded about 86% TVC as compared to fresh, FIG. 14B, about 98% TVC as compared to fresh, FIG. 14C, about 124% as compared to fresh, FIG 14D, about 286% as compared to fresh, and FIG. 14E, about 167% as compared with fresh. The data indicate that overall yield (measured as TVC) from frozen tumor samples on Day 7 is comparable to TVC from fresh tumor samples on Day 11 during early REP.

[0063] Figure 15: Figure 15 depicts the CD8 / CD4 ratio in fresh and frozen tumor samples from 4 patients. Fresh or frozen REP TIL samples were stained with flow cytometry antibodies (CD3-BUV395, CD62LBV421, CD57-PB, CD11c-BV711, CD28-BB515, CD19-BUV563, CCR7-PE, CD123-BV605, CD27PE-CF594, CD14-BV-650, TCRg / d-APC, CD45-PerCP-Cy5.5, CD45RA-A700, CD56-BUV737, CD8-BV786, CD4-PE-Cy7AND CD16-APC-Vio770). The identity of TIL were determined based on TCR a / b+ or g / d+, monocytes (CD14+), NK (CD56+, CD16+, CD56+ / CD16+ and B cells (CD19+) and purity was determined based on CD3+CD45+ cells. Patients M1125 and T6042 demonstrated high CD8 expression, but overall the CD8 / CD4 ratio is comparable between fresh and frozen tumor samples.

[0064] Figure 16: Figures 16A-16H depict CD3+CD45+ expression and other marker expression in the same 4 patients as Figure 15. Figures 16A and 16B depict data for patient M1125; Figures 16C and 16D for patient S13018; Figures 16E and 16F for patient OV8022; and Figures 16G and 16H for patient T6042. The data show that TIL purity is comparable between fresh and frozen tumor samples.

[0065] Figure 17: Figures 17A-17C illustrate expression of CD28, CD27, and CD57 in cells expressing CD4 or CD8 from fresh and frozen tumor samples. Figure 17A is for patient M1125; Figure 17B for patient S13018, and Figure 17C for patient T6042. Data show that the differentiation status between fresh and frozen tumor samples is comparable.

[0066] Figure 18: Figures 18A-18C illustrate memory status in fresh and frozen tumor samples in cells expressing CD4 or CD8. Figure 18A is for patient M1125; Figure 18B for patient S13018, and Figure 18C for patient T6042. Data show that the memory status between fresh and frozen tumor samples is comparable. TN: Naïve (CD45RA+CCR7+), TCM: Central memory (CD45RA-CCR7+), TEM: Effector memory (CD45RA-CCR7-), TEMRA / TEFF: RA+ Effector memory / Effectors (CD45RA+CCR7), Central Memory / Activated cells (CD45RA+CD62L+). Bar graph presented are percentage positive CD45+ / - or CCR7 + / - or CD62L + / - when gated on CD4+ or CD8+.

[0067] Figure 19: Figures 19A-19C illustrate activation / exhaustion markers in fresh and frozen tumor samples in cells expressing CD4 (top panel) or CD8 (bottom panel) in 3 patients: M1125 (FIG. 19A); S13018 (FIG. 19B); and T6042 (FIG. 19C). Activation and exhaustion of REP TIL was determined by multicolor flow cytometry. Tumor tissue was stained with antibodies (CD3- BUV395, PD-1-BV421, 2B4 / CD244-PB, CD8-BB515, CD25-BUV563, BTLA-PE, KLRG1-PE- Dazzle 594, TIM-3-BV650, CD194 / CCR4-APC, CD4-VioGreen, TIGIT-PerCP-eFluor 710, CD183-BV711, CD69-APC-R700, CD95-BUV737, CD127-PE-Cy7, CD103-BV786, LAG-3- APC-eFluor 780) and expression was measured using flow cytometry. Overall, TIL activation / exhaustion status is comparable between fresh and frozen tumor samples.

[0068] Figure 20: Figures 20A-20C illustrate IFNy function in fresh and frozen tumor samples for 3 patients: M1125 (FIG 20A); S13018 (FIG. 20B); and T6042 (FIG. 20C). 1x105 REP TIL were plated in anti-CD3 plates for 24 hours. Culture supernatants were collected and measured for IFNy cytokine using an IFNy Qantikine ELISA kit. Bar graph represent here are Mean + SD of triplicate measurement. Student 't' test was used to calculate statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001. Overall, the data demonstrate that IFNy function is higher in frozen tumor samples.

[0069] Figure 21: Figures 21A-21C illustrate Granzyme B (GzmB) function in fresh and frozen tumor samples for 3 patients: M1125 (FIG 21A); S13018 (FIG. 21B); and T6042 (FIG. 21C). 1x105 REP TIL were plated in anti-CD3 plates for 24 hours. Culture supernatants were collected and measured for Granzyme B using a Duo set ELISA kit. Bar graph represent here are Mean + SD of triplicate measurement. Student 't' test was used to calculate statistical significance. * P <semantics><0.05<annotation encoding="application / x-tex">< 0.05< / annotation>< / semantics>, ** P <semantics><0.01<annotation encoding="application / x-tex">< 0.01< / annotation>< / semantics>, *** P <semantics><0.001<annotation encoding="application / x-tex">< 0.001< / annotation>< / semantics>. Overall, the data demonstrate that Granzyme B (GzmB) function is comparable / higher in frozen tumor samples.

[0070] Figure 22: Figures 22A-22C illustrate CD107A expression in fresh and frozen tumor samples for 3 patients: M1125 (FIG 22A); S13018 (FIG. 22B); and T6042 (FIG. 22C). 2x105 REP TIL were stimulated with or without stimulation of PMA / IO for 2 hours, then stained for CD107A-APC700 and analyzed by flow cytometry. Bar graph represents CD107A positive cells gated on CD8 or CD4. Overall, the data demonstrate that CD107A expression is comparable between fresh and frozen tumor samples.

[0071] Figure 23: Figures 23A-23C illustrate telomere length relative to 1301 cells in fresh and frozen tumor samples for 3 patients: M1125 (FIG 23A); S13018 (FIG. 23B); and T6042 (FIG. 23C). Flow-FISH was performed using Dako / Agilent Pathology Solutions (Telomere PNA) Kit / FITC for Flow Cytometry) kit and the manufacturer's instructions were followed to measure the average length of the Telomere repeat. 1301 T-cell leukemia cell line (SigmaAldrich, St. Louis, MO)) was used as an internal reference standard in each assay. Individual TIL were counted and mixed with 1301 cells at a 1:1 cell ratio. <semantics>2×106<annotation encoding="application / x-tex">2 \times 10^6< / annotation>< / semantics> TIL were mixed with <semantics>2×106<annotation encoding="application / x-tex">2 \times 10^6< / annotation>< / semantics> 1301 cells, in situ hybridization was performed using FITC-conjugated Telomere PNA probe (FITC- 00-CCCTAA-CCC-TAA) and analyzed using Flow cytometry (BD canto II). Telomere fluorescence of the test sample was expressed as a percentage of the geometric mean fluorescence (fl) of the 1301 cells per the following formula: Relative telomere length = [(mean FITC fl test cells w / probe-mean FITC fl test cells w / o probe) × DNA index of 1301 cells × 100] / [(mean FITC fl 1301 cells w / probe – mean FITC fl 1301 cells w / o probe) × DNA index of test cells. The data indicate that telomere length is comparable or better in frozen tumor samples than fresh tumor samples.

[0072] Figure 24: Figure 24 is a flow chart illustrating certain embodiments of the expansion process for TILs from fresh and frozen tumor fragments: fresh method ("GEN2"), early-REP method 1 ("ER-1"), and early-REP method 2 ("ER-2").

[0073] Figure 25: Figure 25 illustrates structures I-A and I-B as comprising three linearly- linked TNFRSF binding domains derived from e.g., 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein through IgG1-Fc (including CH3 and CH2 domains), and is then used to link two of the trivalent proteins together through disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonist capable of bringing together the intracellular signaling domains of the six receptors and signaling proteins to form a signaling complex.

[0074] SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

[0075] SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

[0076] SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2 protein.

[0077] SEQ ID NO:4 is the amino acid sequence of aldesleukin.

[0078] SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4 protein.

[0079] SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7 protein.

[0080] SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15 protein.

[0081] SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21 protein.

[0082] SEQ ID NO:9 is the amino acid sequence of human 4-1BB.

[0083] SEQ ID NO:10 is the amino acid sequence of murine 4-1BB.

[0084] SEQ ID NO:11 is the heavy chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0085] SEQ ID NO:12 is the light chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0086] SEQ ID NO:13 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0087] SEQ ID NO:14 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0088] SEQ ID NO:15 is the heavy chain CDRI for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0089] SEQ ID NO:16 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0090] SEQ ID NO:17 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0091] SEQ ID NO:18 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0092] SEQ ID NO:19 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0093] SEQ ID NO:20 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0094] SEQ ID NO:21 is the heavy chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0095] SEQ ID NO:22 is the light chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0096] SEQ ID NO:23 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0097] SEQ ID NO:24 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0098] SEQ ID NO:25 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0099] SEQ ID NO:26 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[00100] SEQ ID NO:27 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[00101] SEQ ID NO:28 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[00102] SEQ ID NO:29 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[00103] SEQ ID NO:30 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[00104] SEQ ID NO:31 is an Fc domain for a TNFRSF agonist fusion protein.

[00105] SEQ ID NO:32 is a linker for a TNFRSF agonist fusion protein.

[00106] SEQ ID NO:33 is a linker for a TNFRSF agonist fusion protein.

[00107] SEQ ID NO:34 is a linker for a TNFRSF agonist fusion protein.

[00108] SEQ ID NO:35 is a linker for a TNFRSF agonist fusion protein.

[00109] SEQ ID NO:36 is a linker for a TNFRSF agonist fusion protein.

[00110] SEQ ID NO:37 is a linker for a TNFRSF agonist fusion protein.

[00111] SEQ ID NO:38 is a linker for a TNFRSF agonist fusion protein.

[00112] SEQ ID NO:39 is a linker for a TNFRSF agonist fusion protein.

[00113] SEQ ID NO:40 is a linker for a TNFRSF agonist fusion protein.

[00114] SEQ ID NO:41 is a linker for a TNFRSF agonist fusion protein.

[00115] SEQ ID NO:42 is an Fc domain for a TNFRSF agonist fusion protein.

[00116] SEQ ID NO:43 is a linker for a TNFRSF agonist fusion protein.

[00117] SEQ ID NO:44 is a linker for a TNFRSF agonist fusion protein.

[00118] SEQ ID NO:45 is a linker for a TNFRSF agonist fusion protein.

[00119] SEQ ID NO:46 is a 4-1BB ligand (4-1BBL) amino acid sequence.

[00120] SEQ ID NO:47 is a soluble portion of 4-1BBL polypeptide.

[00121] SEQ ID NO:48 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4-1-1 version 1.

[00122] SEQ ID NO:49 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1-1 version 1.

[00123] SEQ ID NO:50 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4-1-1 version 2.

[00124] SEQ ID NO:51 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1-1 version 2.

[00125] SEQ ID NO:52 is a heavy chain variable region (VH) for the 4-1BB agonist antibody H39E3-2.

[00126] SEQ ID NO:53 is a light chain variable region (VL) for the 4-1BB agonist antibody H39E3-2.

[00127] SEQ ID NO:54 is the amino acid sequence of human OX40.

[00128] SEQ ID NO:55 is the amino acid sequence of murine OX40.

[00129] SEQ ID NO:56 is the heavy chain for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00130] SEQ ID NO:57 is the light chain for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00131] SEQ ID NO:58 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00132] SEQ ID NO:59 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00133] SEQ ID NO:60 is the heavy chain CDRl for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00134] SEQ ID NO:61 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00135] SEQ ID NO:62 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00136] SEQ ID NO:63 is the light chain CDR1 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00137] SEQ ID NO:64 is the light chain CDR2 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00138] SEQ ID NO:65 is the light chain CDR3 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[00139] SEQ ID NO:66 is the heavy chain for the OX40 agonist monoclonal antibody 11D4.

[00140] SEQ ID NO:67 is the light chain for the OX40 agonist monoclonal antibody 11D4.

[00141] SEQ ID NO:68 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 11D4.

[00142] SEQ ID NO:69 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 11D4.

[00143] SEQ ID NO:70 is the heavy chain CDRI for the OX40 agonist monoclonal antibody 11D4.

[00144] SEQ ID NO:71 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 11D4.

[00145] SEQ ID NO:72 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 11D4.

[00146] SEQ ID NO:73 is the light chain CDR1 for the OX40 agonist monoclonal antibody 11D4.

[00147] SEQ ID NO:74 is the light chain CDR2 for the OX40 agonist monoclonal antibody 11D4.

[00148] SEQ ID NO:75 is the light chain CDR3 for the OX40 agonist monoclonal antibody 11D4.

[00149] SEQ ID NO:76 is the heavy chain for the OX40 agonist monoclonal antibody 18D8.

[00150] SEQ ID NO:77 is the light chain for the OX40 agonist monoclonal antibody 18D8.

[00151] SEQ ID NO:78 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 18D8.

[00152] SEQ ID NO:79 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 18D8.

[00153] SEQ ID NO:80 is the heavy chain CDRl for the OX40 agonist monoclonal antibody 18D8.

[00154] SEQ ID NO:81 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 18D8.

[00155] SEQ ID NO:82 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 18D8.

[00156] SEQ ID NO:83 is the light chain CDR1 for the OX40 agonist monoclonal antibody 18D8.

[00157] SEQ ID NO:84 is the light chain CDR2 for the OX40 agonist monoclonal antibody 18D8.

[00158] SEQ ID NO:85 is the light chain CDR3 for the OX40 agonist monoclonal antibody 18D8.

[00159] SEQ ID NO:86 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu119-122.

[00160] SEQ ID NO:87 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu119-122.

[00161] SEQ ID NO:88 is the heavy chain CDRl for the OX40 agonist monoclonal antibody Hu119-122.

[00162] SEQ ID NO:89 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu119-122.

[00163] SEQ ID NO:90 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu119-122.

[00164] SEQ ID NO:91 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu119-122.

[00165] SEQ ID NO:92 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu119-122.

[00166] SEQ ID NO:93 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu119-122.

[00167] SEQ ID NO:94 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu106-222.

[00168] SEQ ID NO:95 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu106-222.

[00169] SEQ ID NO:96 is the heavy chain CDRl for the OX40 agonist monoclonal antibody Hu106-222.

[00170] SEQ ID NO:97 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu106-222.

[00171] SEQ ID NO:98 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu106-222.

[00172] SEQ ID NO:99 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu106-222.

[00173] SEQ ID NO:100 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu106-222.

[00174] SEQ ID NO:101 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu106-222.

[00175] SEQ ID NO:102 is an OX40 ligand (OX40L) amino acid sequence.

[00176] SEQ ID NO:103 is a soluble portion of OX40L polypeptide.

[00177] SEQ ID NO: 104 is an alternative soluble portion of OX40L polypeptide.

[00178] SEQ ID NO:105 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 008.

[00179] SEQ ID NO: 106 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 008.

[00180] SEQ ID NO:107 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 011.

[00181] SEQ ID NO:108 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 011.

[00182] SEQ ID NO:109 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 021.

[00183] SEQ ID NO:110 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 021.

[00184] SEQ ID NO:111 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 023.

[00185] SEQ ID NO:112 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 023.

[00186] SEQ ID NO:113 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[00187] SEQ ID NO:114 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[00188] SEQ ID NO:115 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[00189] SEQ ID NO:116 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[00190] SEQ ID NO:117 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[00191] SEQ ID NO:118 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[00192] SEQ ID NO:119 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[00193] SEQ ID NO:120 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[00194] SEQ ID NO:121 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[00195] SEQ ID NO:122 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.

[00196] SEQ ID NO:123 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[00197] SEQ ID NO:124 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.

[00198] SEQ ID NO:125 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.

[00199] SEQ ID NO:126 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.

[00200] SEQ ID NO:127 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[00201] SEQ ID NO:128 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[00202] SEQ ID NO:129 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor nivolumab.

[00203] SEQ ID NO:130 is the light chain variable region (VL) amino acid sequence of the PD- 1 inhibitor nivolumab.

[00204] SEQ ID NO:131 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[00205] SEQ ID NO:132 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[00206] SEQ ID NO:133 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[00207] SEQ ID NO:134 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[00208] SEQ ID NO:135 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[00209] SEQ ID NO:136 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[00210] SEQ ID NO:137 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00211] SEQ ID NO:138 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00212] SEQ ID NO:139 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00213] SEQ ID NO:140 is the light chain variable region (VL) amino acid sequence of the PD- 1 inhibitor pembrolizumab.

[00214] SEQ ID NO:141 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00215] SEQ ID NO:142 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00216] SEQ ID NO:143 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00217] SEQ ID NO:144 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00218] SEQ ID NO:145 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00219] SEQ ID NO:146 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[00220] SEQ ID NO:147 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[00221] SEQ ID NO:148 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[00222] SEQ ID NO:149 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor durvalumab.

[00223] SEQ ID NO:150 is the light chain variable region (VL) amino acid sequence of the PD- L1 inhibitor durvalumab.

[00224] SEQ ID NO:151 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[00225] SEQ ID NO:152 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[00226] SEQ ID NO:153 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[00227] SEQ ID NO:154 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[00228] SEQ ID NO:155 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[00229] SEQ ID NO:156 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[00230] SEQ ID NO:157 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[00231] SEQ ID NO:158 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[00232] SEQ ID NO:159 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor avelumab.

[00233] SEQ ID NO:160 is the light chain variable region (VL) amino acid sequence of the PD- L1 inhibitor avelumab.

[00234] SEQ ID NO:161 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[00235] SEQ ID NO:162 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[00236] SEQ ID NO:163 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[00237] SEQ ID NO:164 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[00238] SEQ ID NO:165 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[00239] SEQ ID NO:166 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[00240] SEQ ID NO:167 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00241] SEQ ID NO:168 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00242] SEQ ID NO:169 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00243] SEQ ID NO:170 is the light chain variable region (VL) amino acid sequence of the PD- L1 inhibitor atezolizumab.

[00244] SEQ ID NO:171 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00245] SEQ ID NO:172 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00246] SEQ ID NO:173 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00247] SEQ ID NO:174 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00248] SEQ ID NO:175 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[00249] SEQ ID NO:176 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab. DETAILED DESCRIPTION OF THE INVENTION

[00250] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Definitions

[00251] The term "frozen" means that the tissue, cellular composition, or other composition, has become hard or rigid as the result of exposure to cold. The term as used herein encompasses the concept of cryopreservation, which is a process of storing cells, tissue, etc., at very low temperatures wherein the stored cells, tissue, etc., maintain their viability.

[00252] The term "cryostorage" means the keeping of already frozen compositions at very low temperatures wherein the stored cells, tissue, etc., maintain their viability.

[00253] The terms "flash freezing," "snap freezing," "flash frozen," and "snap frozen" mean causing to become frozen extremely rapidly, for non-limiting example, in less than about two minutes.

[00254] The term "LOVO cell processing system" and "LOVO" refers to the cell processing system manufactured by Fresenius Kabi USA, LLC. These two terms also refer to any instrument or device manufactured by any vendor that can pump a solution comprising cells through a membrane or filter such as a spinning membrane or spinning filter in a sterile and / or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without pelletization. In some embodiments, such a cell harvester and / or cell processing system can perform cell separation, washing, fluid-exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[00255] The term "fungin" refers to the antifungal reagent Fungin™ sold by InvitroGen, San Diego, CA, USA, (catalog numbers ant-fn-1 and ant-fn-2). Fungin is a soluble formulation of pimaricin, CAS 7681-93-8. As used herein, "fungin" encompasses any commercial formulation of pimaricin or natamycin.

[00256] The term "fungizone" is a trademark of E. R. Squibb and Sons, LLC, and referes to the antimycotic amphotericin B, CAS 1397-89-3. Amphotericin B is commercially available, for example from SIGMA-Aldrich, St. Louis, MO, USA, (catalog number A2942, as a 250 µg / mL solution in deionized water). As used herein, "fungizone" encompasses any commercial formulation of amphotericin B.

[00257] The term "physiologically buffered isotonic saline solution" means any one of the many such salt solutions known to the skilled artisan wherein the solution is made to a physiological pH and isotonic salt concentration. In the art, these are commonly referred to as balanced salt solutions. Without limitation such physiologically buffered isotonic saline solution may comprise Hank's Balanced Salt Solution ("HBSS"), Tris-buffered saline ("TBS"), Phosphate buffered Saline ("PBS"), or Dulbecco's Phosphate Buffered Saline ("DPBS" or "dPBS").

[00258] The term "in vivo" refers to an event that takes place in a subject's body.

[00259] The term "in vitro" refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[00260] The term "ex vivo" refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject's body. Aptly, the cell, tissue and / or organ may be returned to the subject's body in a method of surgery or treatment.

[00261] The term "rapid expansion" means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are outlined below.

[00262] By "tumor infiltrating lymphocytes" or "TILs" herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations can include genetically modified TILs.

[00263] By "population of cells" (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from <semantics>1×106<annotation encoding="application / x-tex">1 \times 10^6< / annotation>< / semantics> to <semantics>1×1010<annotation encoding="application / x-tex">1 \times 10^{10}< / annotation>< / semantics> in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly <semantics>1×108<annotation encoding="application / x-tex">1 \times 10^8< / annotation>< / semantics> cells. REP expansion is generally done to provide populations of <semantics>1.5×109<annotation encoding="application / x-tex">1.5 \times 10^9< / annotation>< / semantics> to <semantics>1.5×1010<annotation encoding="application / x-tex">1.5 \times 10^{10}< / annotation>< / semantics> cells for infusion.

[00264] By "cryopreserved TILs" herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored at a temperature in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity and the avoidance of doubt, "cryopreserved TILs" are distinguishable from frozen tissue samples which may be used as a source of primary TILs.

[00265] By "thawed cryopreserved TILs" herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.

[00266] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.

[00267] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such media can include media comprising 5% v / v DMSO to 10% v / v DMSO; such media can also include media comprising 7% v / v DMSO to 10% v / v DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, as well as combinations thereof. The term "CS10" refers to a cryopreservation medium which is obtained from Stemcell Technologies or from Biolife Solutions. The CS10 medium may be referred to by the trade name "CryoStor® CS10". The CS10 medium is a serum-free, animal component-free medium which comprises DMSO.

[00268] The term "central memory T cell" refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.

[00269] The term "effector memory T cell" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.

[00270] The term "closed system" refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. Closed systems include, for example, but are not limited to closed G- containers. Once a tumor fragment is added to the closed system, the system is not opened to the outside environment until the TILs are ready to be administered to the patient.

[00271] The terms "fragmenting," "fragment," and "fragmented," as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.

[00272] The terms "peripheral blood mononuclear cells" and "PBMCs" refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[00273] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are 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.

[00274] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT- 3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706. TABLE 1. Amino acid sequences of muromonab. [Image disponible dans le document PDF, Image available in the PDF document] TABLE 2. Amino acid sequences of interleukins. [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document]

[00275] 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 thereof. IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 Al. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4902,502. Formulations of IL-2 suitable for use in the invention are described in U.S. Patent No. 6,706,289.

[00276] 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 and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naïve helper T cells (Th0 cells) to 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, and induces class switching to IgE and IgG1 expression from B cells. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15) recombinant protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the invention is given in Table 2 (SEQ ID NO:5).

[00277] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

[00278] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32. IL-15 shares <semantics>β<annotation encoding="application / x-tex">\beta< / annotation>< / semantics> and <semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics> 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 mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:7).

[00279] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc., 13:379-95 (2014). IL-21 is primarily produced by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

[00280] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro, in vivo, and ex vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[00281] A "therapeutic effect" as that term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[00282] "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 inert 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 described compositions and methods.

[00283] When ranges are used herein to describe, for example, physical or chemical properties such as molecular weight or chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Use of the term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary. The variation is typically from 0% to 15%, preferably from 0% to 10%, more preferably from 0% to 5% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes those embodiments such as, for example, an embodiment of any composition of matter, method or process that "consist of" or "consist essentially of" the described features.

[00284] Compounds of the invention also include antibodies. The terms "antibody" and its plural form "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. An "antibody" further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions of an antibody may be further subdivided into regions of hypervariability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes. The constant regions of the antibodies 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.

[00285] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-33, ST2, CD20, PD-1, PD-L1, or PD- L2). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment (Ward et al., Nature, 1989, 341, 544-546), which may consist of a VH or a VL domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as 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 within the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[00286] The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[00287] 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 one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[00288] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[00289] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. In mammals, there are five antibody isotypes: IgA, IgD, IgG, IgM and IgE. In humans, there are four subclasses of the IgG isotype: IgG1, IgG2, IgG3 and IgG4, and two subclasses of the IgA isotype: IgA1 and IgA2.

[00290] The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

[00291] The term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate", "ADC," or "immunoconjugate" refers to an antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a bacterial toxin, a cytotoxic drug or a radionuclide-containing toxin. Toxic moieties can be conjugated to antibodies of the invention using methods available in the art.

[00292] The terms "humanized antibody," "humanized antibodies," and "humanized" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the 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 optionally also will 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.

[00293] The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[00294] A "diabody" is a small antibody fragment with two antigen-binding sites. The fragments comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., 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.

[00295] The term "glycosylation" refers to a modified derivative of an antibody. An aglycoslated antibody lacks glycosylation. Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Aglycosylation may increase the affinity of the antibody for antigen, as described in U.S. Patent Nos. 5,714,350 and 6,350,861. Additionally, or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, 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 in which to express recombinant antibodies of the invention to thereby produce an antibody 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 the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by the 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 a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Patent Publication WO 03 / 035835 describes a variant CHO cell line, Lec 13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, et al., J. Biol. Chem. 2002, 277, 26733-26740. International Patent Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech. 1999, 17, 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[00296] "Pegylation" refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody. Preferably, the 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" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) 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, as described for example in European Patent Nos. EP 0154316 and EP 0401384.

[00297] The term "biosimilar" means a biological product that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or "biosimilar" medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium 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 anti-CD20 monoclonal antibody is rituximab, an anti-CD20 biosimilar monoclonal antibody approved by drug regulatory authorities with reference to rituximab is a "biosimilar to" rituximab or is a "biosimilar thereof" of rituximab. In Europe, a similar biological or "biosimilar" medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, as amended and therefore in Europe, the biosimilar may be authorised, approved for authorisation or subject of an application for authorisation under Article 6 of Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. The already authorized original biological medicinal product may be referred to as a "reference medicinal product" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and / or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorised by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorised outside the European Economic Area (a non-EEA authorised "comparator") in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" also relates to a biological medicinal product which has been or may be compared to a non-EEA authorised comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and / or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, <semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>, 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and / or truncation which is / are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and / or efficacy of the medicinal product. The biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and / or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorised or considered suitable for authorisation. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorisation as a similar biological product. The term "biosimilar" is also used synonymously by other national and regional regulatory agencies.

[00298] The term "hematological malignancy" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies include, but are not limited to, ALL, CLL, SLL, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term "B cell hematological malignancy" refers to hematological malignancies that affect B cells.

[00299] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. The term "solid tumor cancer" refers to malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment.

[00300] 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, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow infiltrating lymphocytes (MILs).

[00301] The term "microenvironment," as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment.

[00302] In an embodiment, the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the invention. In some embodiments, the population of TILs may be provided wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of TILs according to the present invention. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 27 to 23 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours to physiologic tolerance.

[00303] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as "immunosuppressive conditioning") on the patient prior to the introduction of the rTILs of the invention

[00304] The terms "sequence identity," "percent identity," and "sequence percent identity" 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 nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while 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. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[00305] Certain embodiments of the present invention comprise a variant of an antibody, e.g., an anti-IL-33 or anti-ST2 antibody and / or an anti-CD20 antibody and / or an anti-PD-1 antibody, anti-PD-L1 and / or an anti-PD-L2 antibody. As used herein, the term "variant" encompasses but is not limited to antibodies which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody.

[00306] For the avoidance of doubt, it is intended herein that particular features (for example integers, characteristics, values, uses, diseases, formulae, compounds or groups) described in conjunction with a particular aspect, embodiment or example of the invention are to be understood as applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Thus such features may be used where appropriate in conjunction with any of the definition, claims or embodiments defined herein. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. The invention is not restricted to any details of any disclosed embodiments. The invention extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. Tumor Cryopreservation Methods

[00307] The invention provides a method for cryopreserving tumor tissue for the manufacture of tumor infiltrating lymphocytes (TILs) comprising: (i) fragmenting the tumor tissue; (ii) incubating the fragments in a cryopreservation medium; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

[00308] In an embodiment, the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to about 6 mm. In a preferred embodiment, the approximately spherical fragments have a diameter of about 6 mm. In an embodiment, the approximately spherical fragments have a diameter of about 3 mm.

[00309] In some embodiments, the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 1.5 mm and a longest edge length of about 6 mm. In an embodiment, the tumor tissue is fragmented into generally cubical fragments having edge lengths of between about 1.5 mm and 6 mm. In an embodiment, the generally cubical fragments have edge lengths of about 6 mm. In an embodiment, the generally cubical fragments have edge lengths of about 3 mm.

[00310] In some embodiments, the tissue sample is trimmed to separate non-tumor tissue from tumor tissue.

[00311] In some embodiments, the tumor tissue is from a dissected tumor. In an embodiment, the tumor tissue is from a tumor biopsy. In some embodiments the tumor tissue is from an incisional biopsy. In some embodiments the tumor tissue is from an excisional biopsy. In some embodiments the tumor tissues may be from one or more core needle biopsies.

[00312] In some embodiments, the fresh tumor tissue is trimmed into fragments with a cross section of about 1.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1.5 mm, about 2 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 2 mm, about 2.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 2.5 mm, about 3 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3 mm, about 3.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3.5 mm, about 4 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4 mm, about 4.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4.5 mm, about 5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 5 mm, about 5.5 mm × about 5.5 mm, or about 6 mm × about 6 mm.

[00313] In an embodiment, the tumor tissue is less than twelve hours old. In an embodiment, the tumor tissue is less than eight hours old. In an embodiment, the tumor tissue is less than three, less than two, or less than one-hour old.

[00314] In some embodiments, the cryopreservation medium comprises 2 to 12 % v / v (volume:volume) dimethylsulfoxide (DMSO). In some embodiments the cryopreservation medium comprises 5% v / v DMSO. In some embodiments the cryopreservation medium comprises 10% v / v DMSO. In some embodiments, the cryopreservation medium comprises between 5 and 10 % v / v DMSO. In some embodiments the cryopreservation medium comprises a percentage of DMSO selected from the group consisting of 1% v / v, 2% v / v, 3% v / v, 4% v / v, 5% v / v, 6% v / v, 7% v / v, 8% v / v, 9% v / v, 10% v / v, 11% v / v, 12% v / v, 13% v / v, 14% v / v, and 15% v / v. In some of the foregoing embodiments, the remainder of the cryopreservation medium is an aqueous medium.

[00315] In some embodiments, the cryopreservation medium comprises 2 to 12 % w / w (weight:weight) dimethylsulfoxide (DMSO). In some embodiments the cryopreservation medium comprises 5% w / w DMSO. In some embodiments the cryopreservation medium comprises 10% w / w DMSO. In some embodiments, the cryopreservation medium comprises between 5 and 10 % w / w DMSO. In some embodiments the cryopreservation medium comprises a percentage of DMSO selected from the group consisting of 1% w / w, 2% w / w, 3% w / w , 4% w / w , 5% w / w , 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11% w / w, 12% w / w, 13% w / w, 14% w / w, and 15% w / w. In some of the foregoing embodiments, the remainder of the cryopreservation medium is an aqueous medium.

[00316] In an embodiment, the cryopreservation medium comprises at least one antimicrobial agent. In an embodiment, the at least one antimicrobial agent is gentamicin at a concentration of at least 50 µg / mL. In some embodiments, the at least one antimicriobial agent is penicillin; in some embodiments the at least one antimicriobial agent is streptomycin. In some embodiments the at least one antimicrobial agent is an antifungal agent. In some embodiments the antifungal agent is amphotericin B; in some embodiments the antifungal agent is FunginTM. In some embodiments combinations of antimicriobial agents are used. In some embodiments at least one antifungal agent is used in combination with one or more antibacterial agents.

[00317] In some embodiments, the tumor fragments are incubated in cryopreservation medium for about 20 minutes to about 70 minutes. In some embodiments, the tumor fragments are incubated in cryopreservation medium for about 30 minutes to about 60 minutes. In some embodiments the incubation is at least 10 minutes; at least 20 minutes; at least 25 minutes; at least 30 minutes; at least 35 minutes; at least 40 minutes; at least 45 minutes; at least 50 minutes; at least 55 minutes; at least 60 minutes; or at least 70 minutes. In some embodiments the incubation is about 10 minutes; about 20 minutes; about 25 minutes; about 30 minutes; about 35 minutes; about 40 minutes; about 45 minutes; about 50 minutes; about 55 minutes; about 60 minutes; or about 70 minutes. In some embodiments the incubation is less than 10 minutes; less than 20 minutes; less than 25 minutes; less than 30 minutes; less than 35 minutes; less than 40 minutes; less than 45 minutes; less than 50 minutes; less than 55 minutes; less than 60 minutes; or less than 70 minutes. In some embodiments the incubation time is proportional to tumor fragment density. In some embodiments the incubation time is proportional to tumor fragment surface to volume ratio.

[00318] In some embodiments, the tumor fragments are incubated in a cryopreservation medium at a temperature from about 2°C to about 8°C.

[00319] In some embodiments, the tumor tissue is washed in a physiologically buffered isotonic saline solution. In some embodiments, the washing comprises three serial washes of at least three minutes each, with the physiologically buffered isotonic saline solution replaced after each serial wash. In some embodiments, the physiologically buffered isotonic saline solution comprises Hank's Balance Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline solution comprises tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline solution comprises phosphate buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline solution comprises Dulbecco's phosphate-buffered saline (DPBS). In some embodiments, the physiologically buffered isotonic saline solution in one serial wash may be a different physiologically buffered isotonic saline solution than used in one or more of the other serial washes.

[00320] In an embodiment, the freezing takes place at a temperature in the range from about - 125°C to about -196°C. In an embodiment, the freezing takes place at a temperature in the range of about -140°C to about -185°C. In an embodiment, the freezing takes place at a temperature in the range of about -140°C to about -175°C. In an embodiment, the freezing takes place at a temperature of about -145°C. In some embodiments, the freezing takes place in the vapor phase of liquid nitrogen.

[00321] A problem well-known in the art is to cryopreserve cells or tissues without damaging them during the freezing process. Without being bound by theory, one source of damage during freezing is intracellular ice nucleation resulting in cellular rupture. Muldrew and McGann outline a quantitative theory for this well known and widely recognized difficulty with cellular and, in particular, whole tissue cryopreservation in "The osmotic rupture hypothesis of intracellular freezing injury", Biophysical Journal, 66:532-41 (1994). Acker and McGann further develop the fundamental mechanisms of intracellular ice formation and cellular damage in a later article, "Membrane damage occurs during the formation of intracellular ice," Cryo Letter, 22:241-54 (2001).

[00322] Damage during freezing is detected wherein upon thawing, the tissues have substantially lost their physiological structures or the cells comprising the tissue have substantially lost their viability. Viability may be determined by the fraction of cells introduced into a culture medium compared to the number of such cells that grow or show markers of normal cellular function. Numerous methods are known in the art to identify the fraction of viable cells, for example, and without limitation, dye exclusion tests, such as trypan blue exclusion. See, for example, Strober, Curr. Protoc. Immunol., 2001, Appendix 3B, available at https: / / dx.doi.org / 10.1002 / 0471142735.ima03bs21. Without limitation, viability may also be determined by metabolic activity assays, such as the MTT assay, wherein MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, is metabolized by cellular enzymes into formazan. This enzymatic reaction converts the yellow MTT into purple formazan. See, for example, Berridge et al., Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, 11: 127-152 (2005); Mosmann, "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays," J. Immunol. Methods 65 (1-2): 55-63 (1983).

[00323] Without being bound by theory, slow cooling is hypothesized to produce innocuous intracellular ice, see Acker and McGann, "Protective effect of intracellular ice during freezing?" Cryobiology, 46(2): 197-202 (2003). A conventional approach to achieve freezing without excessive cellular damage or to achieve freezing without significantly reducing cellular viability has been to employ slow freezing rates. Watson et al., U.S. Patent 5,891,617, emphasize this approach, for example claim one, step (c), teaches a very slow rate of cooling: "about -0.3° C. per minute or less." Similarly, Comhaire et al., U.S. Patent 9,938,495, teach "high cell viability after thawing were obtained by slow freezing using a DMSO-free cryopreservation medium" for stem cells.

[00324] Based on these exemplary teachings, the methods and products of the present disclosure are surprising and unexpected. Further the present disclosure, including the examples and the data therein, demonstrate a technical solution to the problem of rapidly and efficiently cryoprotecting tumor tissues, tumor fragments, or tumor specimens, for the use in the manufacture of tumor infiltrating lymphocytes for therapeutic use.

[00325] In some embodiments, the method of cryopreserving tumor tissue for the manufacture of tumor infiltrating lymphocytes (TILs), further comprises a step (iv) storing the frozen fragments at a temperature below at least -130°C. In some embodiments, the frozen fragments are stored in the vapor phase of liquid nitrogen. In some embodiments, the frozen fragments are stored submerged in liquid nitrogen. In some embodiments, the cryopreserved fragments are stored for later manufacture of TILs for autologous therapeutic use.

[00326] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising the steps of: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising incubating the tumor tissue for a period of about 20 minutes to about 70 minutes in a storage medium at a temperature from about 2°C to about 8°C before freezing; (b) thawing the tumor tissue; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 antibody to provide TILs; (d) removing at least a plurality of the TILs; and (e) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00327] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising the steps of: (a) fragmenting a tumor sample; (b) incubating the tumor fragments in a storage medium; (c) storing the tumor fragments in a frozen state; (d) thawing the tumor fragments; (e) treating the tumor fragments in a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 antibody to provide TILs; (f) removing at least a plurality of the TILs; and (g) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00328] In some embodiments, the storage medium comprises 2 to 12 % v / v (volume:volume or volume-to-volume) dimethylsulfoxide (DMSO). In some embodiments the storage medium comprises 5% v / v DMSO. In some embodiments the storage medium comprises 10% v / v DMSO. In some embodiments, the storage medium comprises between 5 and 10 % v / v DMSO. In some embodiments the storage medium comprises a percentage of DMSO selected from the group consisting of 1% v / v, 2% v / v, 3% v / v, 4% v / v, 5% v / v, 6% v / v, 7% v / v, 8% v / v, 9% v / v, <semantics>10% v / v<annotation encoding="application / x-tex">10\% \text{ v / v}< / annotation>< / semantics>, <semantics>11% v / v<annotation encoding="application / x-tex">11\% \text{ v / v}< / annotation>< / semantics>, <semantics>12% v / v<annotation encoding="application / x-tex">12\% \text{ v / v}< / annotation>< / semantics>, <semantics>13% v / v<annotation encoding="application / x-tex">13\% \text{ v / v}< / annotation>< / semantics>, <semantics>14% v / v<annotation encoding="application / x-tex">14\% \text{ v / v}< / annotation>< / semantics>, and <semantics>15% v / v<annotation encoding="application / x-tex">15\% \text{ v / v}< / annotation>< / semantics>. In some of the foregoing embodiments, the remainder of the storage medium is an aqueous medium.

[00329] In some embodiments, the storage medium comprises 2 to 12 % w / w (weight:weight or weight-to-weight) dimethylsulfoxide (DMSO). In some embodiments the storage medium comprises 5% w / w DMSO. In some embodiments the storage medium comprises 10% w / w DMSO. In some embodiments, the storage medium comprises between 5 and 10 % w / w DMSO. In some embodiments the storage medium comprises a percentage of DMSO selected from the group consisting of 1% w / w, 2% w / w, 3% w / w , 4% w / w , 5% w / w , 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11% w / w, 12% w / w, 13% w / w, 14% w / w, and 15% w / w. In some of the foregoing embodiments, the remainder of the storage medium is an aqueous medium.

[00330] In some embodiments, the tumor tissue is incubated for between about 20 minutes and about 70 minutes in a storage medium at a temperature from about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for at least 30 minutes in a storage medium at a temperature from about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for between 30 minutes and about 60 minutes in a storage medium at a temperature from about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 15 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 45 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 75 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for 60 minutes in a storage medium at a temperature from about 2°C to about 8°C before freezing.

[00331] In some embodiments, the storage medium comprises about 5% v / v DMSO and the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature from about 2°C to about 8°C before freezing. In some embodiments, the storage medium comprises about 10% v / v DMSO and the tumor tissue is incubated for at least 30 minutes to about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments the incubation time is proportional to tumor fragment density. In some embodiments the incubation time is proportional to tumor fragment surface to volume ratio. Cryopreserved Tumor Fragments

[00332] In some embodiments, the invention provides a cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by a process comprising the steps of: (i) fragmenting a tumor sample; (ii) incubating the fragments in a cryopreservation medium; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

[00333] In some embodiments, the invention provides a cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by a process comprising the steps of: (i) fragmenting a tumor sample; (ii) incubating the fragments for about 20 minutes to about 70 minutes at a temperature in the range of about 2°C to about 8°C in a cryopreservation medium comprising 10% v / v DMSO; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

[00334] In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 6 mm. In an embodiment, the approximately spherical fragments have a diameter of about 6 mm. In an embodiment, the approximately spherical fragments have a diameter of about 3 mm. In an embodiment, the approximately spherical fragments have a diameter selected from the group consisting of about 20 mm, about 19 mm, about 18 mm, about 17 mm, about 16 mm, about 15 mm, about 14 mm, about 13 mm, about 12 mm, about 11 mm, about 10 mm, about 9 mm, about 8 mm, about 7 mm, about 6 mm, about 5 mm, about 4 mm, about 3 mm, about 2 mm, and about 1 mm.

[00335] In some embodiments, the tumor sample is fragmented into generally rectangular fragments having a shortest edge length of at least 1.5 mm and a longest edge of about 6 mm. In an embodiment, the tumor sample is fragmented into generally cubical fragments having edges with lengths of between about 1.5 mm and 6 mm. In an embodiment, the generally cubical fragments have edges with lengths of about 6 mm. In an embodiment, the generally cubical fragments have edges with lengths of about 3 mm. In an embodiment, the generally cubical fragments have an edge length selected from the group consisting of about 20 mm, about 19 mm, about 18 mm, about 17 mm, about 16 mm, about 15 mm, about 14 mm, about 13 mm, about 12 mm, about 11 mm, about 10 mm, about 9 mm, about 8 mm, about 7 mm, about 6 mm, about 5 mm, about 4 mm, about 3 mm, about 2 mm, and about 1 mm.

[00336] In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 2 mm3 to about 200 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 5 mm3 to about 150 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 25 mm3 to about 150 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 50 mm3 to about 150 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 100 mm3 to about 125 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 50 mm3 to about 75 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 75 mm3 to about 100 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 200 mm3 to about 1000 mm3. In an embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 500 mm3 to about 800 mm3. In an embodiment, the approximately spherical fragments have a diameter selected from the group consisting of about 20 mm, about 19 mm, about 18 mm, about 17 mm, about 16 mm, about 15 mm, about 14 mm, about 13 mm, about 12 mm, about 11 mm, about 10 mm, about 9 mm, about 8 mm, about 7 mm, about 6 mm, about 5 mm, about 4 mm, about 3 mm, about 2 mm, and about 1 mm.

[00337] In some embodiments, the fresh tumor tissue is trimmed into fragments, wherein a fragment has a cross section of about 1.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1.5 mm, about 2 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 2 mm, about 2.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 2.5 mm, about 3 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3 mm, about 3.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3.5 mm, about 4 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4mm, about 4.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4.5 mm, about 5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 5 mm, about 5.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> about 5.5 mm, about 6 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6 mm, about 6.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6.5 mm, about 7 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 7 mm, about 7.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 7.5 mm, about 8 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 8 mm, about 8.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 8.5 mm, about 9 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 9 mm, about 9.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 9.5 mm, about 10 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 10 mm, about 10.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 10.5 mm, about 11 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 11 mm, about 11.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 11.5 mm, about 12 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 12 mm.

[00338] In some embodiments, the fresh tumor tissue is trimmed into fragments, wherein a fragment has a cross-sectional area of about 2 mm2 to about 3 mm2, about 3 mm2 to about 4 mm2, about 4 mm2 to about 5 mm2, about 5 mm2 to about 6 mm2, about 6 mm2 to about 7 mm2, about 7 mm2 to about 8 mm2, about 8 mm2 to about 9 mm2, about 9 mm2 to about 10 mm2, about 10 mm2 to about 11 mm2, about 11 mm2 to about 12 mm2, about 12 mm2 to about 20 mm2, about 20 mm2 to about 50 mm2, about 50 mm2 to about 100 mm2, or about 100 mm2 to about 500 mm2. In some embodiments, the fresh tumor tissue is trimmed into fragments, wherein a fragment has a cross-sectional area of about 1.5 mm2, about 2 mm2, about 2.5 mm2, about 3 mm2, about 3.5 mm2, about 4 mm2, about 4.5 mm2, about 5 mm2, about 5.5 mm2, about 6 mm2, about 6.5 mm2, about 7 mm2, about 7.5 mm2, about 8 mm2, about 8.5 mm2, about 9 mm2, about 9.5 mm2, about 10 mm2, about 10.5 mm2, about 11 mm2, about 11.5 mm2, about 12 mm2, about 20 mm2, about 25 mm2, about 30 mm2, about 40 mm2, about 50 mm2, about 100 mm2, about 200 mm2, about 300 mm2, about 400 mm2, about 500 mm2, about 750 mm2, or about 1000 mm2.

[00339] In some embodiments, the fresh tumor tissue is trimmed into fragments, wherein a fragment has a volume of about 2 mm3 to about 3 mm3, about 3 mm3 to about 4 mm3, about 4 mm3 to about 5 mm3, about 5 mm3 to about 6 mm3, about 6 mm3 to about 7 mm3, about 7 mm3 to about 8 mm3, about 8 mm3 to about 9 mm3, about 9 mm3 to about 10 mm3, about 10 mm3 to about 11 mm3, about 11 mm3 to about 12 mm3, and about 12 mm3 to about 20 mm3. In some embodiments, the fresh tumor tissue is trimmed into fragments, wherein a fragment has a volume of about 1.5 mm3, about 2 mm3, about 2.5 mm3, about 3 mm3, about 3.5 mm3, about 4 mm3, about 4.5 mm3, about 5 mm3, about 5.5 mm3, about 6 mm3, about 6.5 mm3, about 7 mm3, about 7.5 mm3, about 8 mm3, about 8.5 mm3, about 9 mm3, about 9.5 mm3, about 10 mm3, about 10.5 mm3, about 11 mm3, about 11.5 mm3, about 12 mm3, about 20 mm3, about 25 mm3, about 30 mm3, about 40 mm3, about 50 mm3, about 100 mm3, about 200 mm3, about 300 mm3, about 400 mm3, about 500 mm3, about 750 mm3, or about 1000 mm3.

[00340] In some embodiments, the tumor sample is from a dissected tumor. In an embodiment, the tumor sample is from a tumor biopsy. In some embodiments the tumor sample is from an incisional biopsy. In some embodiments the tumor sample is from an excisional biopsy. In some embodiments the tumor samples may be from one or more core needle biopsies.

[00341] In an embodiment, the tumor sample is less than twelve hours old prior to freezing. In an embodiment, the tumor sample is less than eight hours old prior to freezing. In an embodiment, the tumor sample is less than three, less than two, or less than one-hour old prior to freezing.

[00342] In some embodiments, the cryopreservation medium comprises 2 to 12 % v / v (volume:volume or volume-to-volume) dimethylsulfoxide (DMSO). In some embodiments the cryopreservation medium comprises 5% v / v DMSO. In some embodiments the cryopreservation medium comprises 10% v / v DMSO. In some embodiments, the cryopreservation medium comprises between 5 and 10 % v / v DMSO. In some embodiments the cryopreservation medium comprises a percentage of DMSO selected from the group consisting of 1% v / v, 2% v / v, 3% v / v, 4% v / v, 5% v / v, 6% v / v, 7% v / v, 8% v / v, 9% v / v, 10% v / v, 11% v / v, 12% v / v, 13% v / v, 14% v / v, and 15% v / v. In some of the foregoing embodiments, the remainder of the cryopreservation medium is an aqueous medium.

[00343] In some embodiments, the cryopreservation medium comprises 2 to 12 % w / w (weight:weight or weight-to-weight) dimethylsulfoxide (DMSO). In some embodiments the cryopreservation medium comprises 5% w / w DMSO. In some embodiments the storage medium comprises 10% w / w DMSO. In some embodiments, the cryopreservation medium comprises between 5 and 10 % w / w DMSO. In some embodiments the cryopreservation medium comprises a percentage of DMSO selected from the group consisting of 1% w / w, 2% w / w, 3% w / w, 4% w / w, 5% w / w, 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11% w / w, 12% w / w, 13% w / w, 14% w / w, and 15% w / w. In some of the foregoing embodiments, the remainder of the cryopreservation medium is an aqueous medium.

[00344] In an embodiment, the cryopreservation medium comprises at least one antimicrobial agent. In an embodiment, the at least one antimicrobial agent is gentamicin at a concentration of at least 20 µg / mL. In some embodiments, the at least one antimicrobial agent is gentamicin at a concentration of at least 50 µg / mL. In some embodiments, the at least one antimicriobial agent is penicillin; in some embodiments the at least one antimicriobial agent is streptomycin. In some embodiments the at least one antimicrobial agent is an antifungal agent. In some embodiments the antifungal agent is amphotericin B; in some embodiments the antifungal agent is FunginTM. In some embodiments combinations of antimicriobial agents are used. In some embodiments at least one antifungal agent is used in combination with one or more antibacterial agents.

[00345] In some embodiments, the tumor fragments are incubated in cryopreservation medium for about 30 minutes to about 60 minutes. In some embodiments the incubation is at least 10 minutes; at least 20 minutes; at least 25 minutes; at least 30 minutes; about 35 minutes; about 40 minutes; about 45 minutes; about 50 minutes; about 55 minutes; or about 60 minutes. In some embodiments the incubation time is proportional to tumor fragment density. In some embodiments the incubation time is proportional to tumor fragment surface to volume ratio.

[00346] In some embodiments, the tumor fragments are incubated in cryopreservation medium at a temperature from about 2°C to about 8°C.

[00347] In some embodiments, the tumor sample is washed in a physiologically buffered isotonic saline solution. In some embodiments, the washing comprises three serial washes of at least three minutes each, with the physiologically buffered isotonic saline solution replaced after each serial wash. In some embodiments, the physiologically buffered isotonic saline solution comprises Hank's Balance Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline solution comprises tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline solution comprises phosphate buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline solution comprises Dulbecco's phosphate-buffered saline (DPBS). In some embodiments, the physiologically buffered isotonic saline solution in one serial wash may be a different physiologically buffered isotonic saline solution than used in one or more of the other serial washes.

[00348] In an embodiment, the freezing takes place at a temperature in the range from about - 125°C to about -180°C. In an embodiment, the freezing takes place at a temperature in the range of about -140°C to about -175°C. In an embodiment, the freezing takes place at a temperature of about -145°C. In some embodiments, the freezing takes place in the vapor phase of liquid nitrogen.

[00349] In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is one. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is two. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is three. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is four. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is five. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is six. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is seven. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is eight. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is nine. In an embodiment, the number of tumor fragments used according to any of the embodiments above or herein is ten. Manufacturing TILs from Cryopreserved Tumor Fragments

[00350] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from a cryopreserved tumor fragment into a therapeutic population of TILs, the method comprising the following steps: (i) obtaining a first population of TILs from a cryopreserved tumor fragment; (ii) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally OKT-3 to produce a second population of TILs; and (iii) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is at least 50-fold or 100-fold greater in number than the second population of TILs, and wherein the second expansion is performed for at least 14 days in order to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs.

[00351] The invention provides a method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy, the method comprising: (a) culturing a cryopreserved tumor fragment in a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 antibody to provide TILs; (b) expanding the TILs in a second cell culture medium comprising irradiated feeder cells, OKT-3 antibody, and IL-2 to provide an expanded number of TILs; and, (c) optionally cryopreserving the expanded number of TILs.

[00352] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising the steps of: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising incubating the tumor tissue for about 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing; (b) thawing the tumor tissue; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (d) removing at least a plurality of the TILs; and (e) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00353] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising the steps of: (a) fragmenting a tumor sample; (b) incubating the tumor fragments in a storage medium for about 30 minutes at a temperature in the range of about 2°C to about 8°C; (c) storing the tumor fragments in a frozen state; (d) thawing the tumor fragments; (e) treating the tumor fragments in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (f) removing at least a plurality of the TILs; and (g) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00354] In some embodiments, the storage medium comprises 2% v / v DMSO to 12 % v / v dimethylsulfonoxide (DMSO). In some embodiments the storage medium comprises 5% v / v DMSO. In some embodiments the storage medium comprises 10% v / v DMSO. In some embodiments, the storage medium comprises between about 5% v / v DMSO and 10 % v / v DMSO.

[00355] In some embodiments, the tumor tissue is incubated for at least 30 minutes in a storage medium at a temperature from about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for between 30 minutes and about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 45 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing. In some embodiments, the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C before freezing.

[00356] In some embodiments, the tumor tissue is incubated in cryopreservation medium for about 20 minutes to about 70 minutes. In some embodiments, the tumor tissue is incubated in cryopreservation medium for about 30 minutes to about 60 minutes. In some embodiments the incubation is at least 10 minutes; at least 20 minutes; at least 25 minutes; at least 30 minutes; about 35 minutes; about 40 minutes; about 45 minutes; about 50 minutes; about 55 minutes; or about 60 minutes. In some embodiments the incubation time is proportional to tumor fragment density. In some embodiments the incubation time is proportional to tumor fragment surface to volume ratio.

[00357] In some embodiments, a tumor fragment with a cross section of about 1.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1.5 mm is incubated in cryopreservation medium for about 20 minutes, for about 30 minutes, for about 35 minutes, for about 40 minutes, or for less than about 60 minutes. In some embodiments, a tumor fragment with a cross section of about 2 mm × 2 mm is incubated in cryopreservation medium for about 20 minutes, for about 30 minutes, for about 35 minutes, for about 40 minutes, for about 45 minutes, for about 50 minutes, or for about 60 minutes. In some embodiments, a tumor fragment with a cross section of about 3 mm × 3 mm is incubated in cryopreservation medium for at least 20 minutes, for about 30 minutes, for about 35 minutes, for about 40 minutes, for about 45 minutes, for about 50 minutes, or for about 60 minutes. In some embodiments, a tumor fragment with a cross section of about 4 mm × 4 mm is incubated in cryopreservation medium for about 30 minutes, for about 35 minutes, for about 40 minutes, for about 45 minutes, for about 50 minutes, for about 60 minutes, or for about 70 minutes. In some embodiments, a tumor fragment with a cross section of about 5 mm × 5 mm is incubated in cryopreservation medium for about 30 minutes, for about 35 minutes, for about 40 minutes, for about 45 minutes, for about 50 minutes, for about 60 minutes, or for about 70 minutes. In some embodiments, a tumor fragment with a cross section of about 6 mm × 6 mm is incubated in cryopreservation medium for at least 20 minutes, for about 30 minutes, for about 40 minutes, for about 45 minutes, for about 50 minutes, for about 60 minutes, or for about 70 minutes.

[00358] In some embodiments, the storage medium comprises about 5% v / v DMSO and the tumor tissue is incubated for about 60 minutes in a storage medium at a temperature in the range from about 2°C to about 8°C before freezing. In some embodiments, the storage medium comprises about 10% v / v DMSO and the tumor tissue is incubated for at least about 30 minutes to about 60 minutes in a storage medium at a temperature in the range from about 2°C to about 8°C before freezing.

[00359] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises incubating cryopreserved tumor fragments in a culture medium comprising IL-2, wherein the number of TILs are expanded. In some embodiments, the number of TILs is expanded 100-fold. In some embodiments, the number of TILs is expanded 150-fold. In some embodiments, the number of TILs is expanded 200-fold. In some embodiments, the number of TILs is expanded 250-fold. In some embodiments, the number of TILs is expanded about 300- fold. In some embodiments, the number of TILs is expanded about 350-fold. In some embodiments, the number of TILs is expanded about 400-fold. In some embodiments, the number of TILs is expanded about 450-fold. In some embodiments, the number of TILs is expanded about 500-fold. In some embodiments, the number of TILs is expanded about 550- fold. In some embodiments, the number of TILs is expanded about 600-fold. In some embodiments, the number of TILs is expanded about 700-fold. In some embodiments, the number of TILs is expanded about 750-fold. In some embodiments, the number of TILs is expanded about 800-fold. In some embodiments, the number of TILs is expanded about 850- fold. In some embodiments, the number of TILs is expanded about 900-fold. In some embodiments, the number of TILs is expanded about 1000-fold. In some embodiments, the number of TILs is expanded about 1200-fold. In some embodiments, the number of TILs is expanded about 1500-fold. In some embodiments, the number of TILs is expanded about 1600- fold. In some embodiments, the number of TILs is expanded about 2000-fold. In some embodiments, the number of TILs is expanded about 2100-fold. In some embodiments, the number of TILs is expanded about 2200-fold. In some embodiments, the number of TILs is expanded about 2500-fold. In some embodiments, the number of TILs is expanded greater than 2500-fold.

[00360] In some embodiments, a method for manufacturing TILs from frozen tumor tissue comprises: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (iv) freezing the vessel; and (v) storing the vessel such that the vessel remains frozen; (b) thawing the vessel; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (d) removing at least a plurality of the TILs; and (e) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00361] In some embodiments, a method for manufacturing TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) freezing the vessel; (f) storing the vessel such that the vessel remains frozen; (g) thawing the vessel; (h) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (i) removing at least a plurality of the TILs; and (j) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00362] In some embodiments, the method of manufacturing TILs from a cryopreserved tumor fragment, further comprises addition to OKT-3 to the first culture medium.

[00363] In some embodiments, the fresh tumor tissue is trimmed to between 1.5 mm and 6 mm in diameter and between 1.5 mm and 6 mm in thickness. In yet other embodiments, the fresh tumor tissue is trimmed to about 6 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6 mm. In some embodiments, the fresh tumor tissue is trimmed into fragments with a cross section of about 1.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1.5 mm, about 2 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 2 mm, about 2.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 2.5 mm, about 3 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3 mm, about 3.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3.5 mm, about 4 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4 mm, about 4.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4.5 mm, about 5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 5 mm, about 5.5 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> about 5.5 mm, or about 6 <semantics>mm×6mm<annotation encoding="application / x-tex">mm \times 6 mm< / annotation>< / semantics>.

[00364] In some embodiments, the storage medium further comprises an antibacterial agent. In some embodiments the antibacterial agent is gentamicin. In some embodiments the storage medium comprises gentamicin at a concentration of at least 20 µg / mL. In some embodiments the storage medium comprises gentamicin at a concentration of at least 50 µg / mL. In some embodiments the storage medium comprises an antibacterial agent, an antifungal agent, and combinations thereof. In some embodiments, the antifungal agent is amphotericin B and is present from about 0.25 µg / mL to about 2.5 µg / mL. In some embodiments, the antifungal agent is FunginTM and is present from about 5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>g / mL to about 50 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>g / mL.

[00365] In some embodiments, the tumor tissue is washed in a physiologically buffered isotonic saline solution. In some embodiments, the washing comprises three serial washes of at least three minutes each, with the physiologically buffered isotonic saline solution replaced after each serial wash. In some embodiments, the physiologically buffered isotonic saline solution comprises Hank's Balance Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline solution comprises tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline solution comprises phosphate buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline solution comprises Dulbecco's phosphate-buffered saline (DPBS). In some embodiments, the physiologically buffered isotonic saline solution in one serial wash may be a different physiologically buffered isotonic saline solution than used in one or more of the other serial washes.

[00366] In some embodiments the thawing step comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[00367] In some embodiments, the freezing step comprises freezing the vessel at about -125°C to about -195°C. In some embodiments, the vessel is frozen at a temperature from about -125°C to about -150°C; in some embodiments the vessel is frozen at a temperature from about -125°C to about -145°C; in yet further embodiments, the vessel is frozen at a temperature from about - 135°C.

[00368] In some embodiments, the method is performed with a fresh tumor sample from a human subject.

[00369] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC) tissue, and HPV-positive tumor tissue.

[00370] In some embodiments, the method further comprises a split between the first expansion (or first culture) step and the second expansion (or second culture) step. In an embodiment, the split occurs on about day 16. In some embodiments, the first culture step is complete in about 11 days. In other embodiments, the first expansion (or first culture) step is complete in about 7 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 7 days and the second expansion (or second culture) step is complete in about 14 days. In some embodiments, steps (c) through (e) are complete in about 22 days. In yet other embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are complete in about 21 days; in some embodiments steps (c) through (e) or steps (h) through (j), as applicable, are complete in about 20 days; and in some embodiments steps (c) through (e) or steps (h) through (j), as applicable, are complete in about 16 days.

[00371] In some embodiments, the first culture medium comprises OKT-3.

[00372] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof, such methods comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 30 minutes; (iv) freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (b) thawing the tumor tissue; (c) adding the tumor tissue into a closed system; (d) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein 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 population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the therapeutic population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested TIL population from step (g) using a cryopreservation process; and (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject.

[00373] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof are provided, such methods comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising: (a) obtaining tumor tissue from the subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 30 minutes; (e) freezing the vessel using the vapor phase of liquid nitrogen; (f) storing the vessel at the vapor phase of liquid nitrogen temperature; (g) thawing the tumor tissue; (h) adding the tumor tissue into a closed system; (i) performing in the closed system a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (i) to step (j) occurs without opening the system; (k) harvesting the therapeutic population of TILs obtained from step (j), wherein the transition from step (j) to step (k) occurs without opening the system; (1) transferring the therapeutic of TILs harvested in step (k) to an infusion bag, wherein the transfer from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (1) using a cryopreservation process; (n) thawing the therapeutic population of TILs in the infusion bag from step (m); and (o) administering a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (n) to the subject.

[00374] In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma, endometrial cancer, thyroid cancer, colorectal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, melanoma, refractory melanoma, metastatic melanoma, and non-small cell lung carcinoma. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment.

[00375] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising incubating the tumor tissue for 30 minutes in a storage medium at 2°C - 8°C before freezing; (c) thawing the tumor tissue; (d) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (e) removing at least a plurality of the TILs; and (f) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00376] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) incubating the tumor tissue for 30 minutes in a storage medium at 2°C - 8°C; (c) storing the tumor tissue in a frozen state; (d) thawing the tumor tissue; (e) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (f) removing at least a plurality of the TILs; and (g) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00377] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (1) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 30 minutes; (iv) freezing the vessel; and (v) storing the vessel such that the vessel remains frozen; (c) thawing the vessel; (d) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (e) removing at least a plurality of the TILs; and (f) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00378] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 30 minutes; (e) freezing the vessel; (f) storing the vessel such that the vessel remains frozen; (g) thawing the vessel; (h) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (i) removing at least a plurality of the TILs; and (j) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00379] In some embodiments, the tumor tissue is washed in a physiologically buffered isotonic saline solution. In some embodiments, the washing comprises three serial washes of at least three minutes each, with the physiologically buffered isotonic saline solution replaced after each serial wash. In some embodiments, the physiologically buffered isotonic saline solution comprises Hank's Balance Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline solution comprises tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline solution comprises phosphate buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline solution comprises Dulbecco's phosphate-buffered saline (DPBS). In some embodiments, the physiologically buffered isotonic saline solution in one serial wash may be a different physiologically buffered isotonic saline solution than used in one or more of the other serial washes.

[00380] In some embodiments the thawing step comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[00381] In some embodiments, the freezing step comprises freezing the vessel at about -125°C to about -195°C. In some embodiments, the vessel is frozen at a temperature from about -125°C to about -150°C; in some embodiments the vessel is frozen at a temperature from about -125°C to about -145°C; in yet further embodiments, the vessel is frozen at a temperature of about - 135°C.

[00382] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC) tissue, and HPV-positive tumor tissue.

[00383] In some embodiments, the method further comprises a split between the first expansion (or first culture) step and the second expansion (or second culture) step. In an embodiment, the split occurs on about day 16. In some embodiments, the first culture step is complete in about 11 days. In other embodiments, the first expansion (or first culture) step is complete in about 7 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 7 days and the second expansion (or second culture) step is complete in about 14 days. In some embodiments, steps (d) through (f) or steps (h) through (j), as applicable, are complete in about 22 days. In yet other embodiments, steps (d) through (f) or steps (h) through (j), as applicable, are complete in about 21 days; in some embodiments steps (d) through (f) or steps (h) through (j), as applicable, are complete in about 20 days; and in some embodiments steps (d) through (f) or steps (h) through (j), as applicable, are complete in about 16 days.

[00384] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof, such methods comprising: (a) obtaining tumor tissue from the subject, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 to about 70 minutes; (iv) freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (c) thawing the tumor tissue; (d) adding the tumor tissue into a closed system; (e) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (e) to step (f) occurs without opening the system; (g) harvesting the therapeutic population of TILs obtained from step (f), wherein the transition from step (f) to step (g) occurs without opening the system; (h) transferring the harvested TIL population from step (g) to an infusion bag, wherein the transfer from step (g) to (h) occurs without opening the system; (i) cryopreserving the infusion bag comprising the harvested TIL population from step (h) using a cryopreservation process; and (1) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (i) to the subject.

[00385] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof, such methods comprising: (a) obtaining tumor tissue from the subject, wherein the tumor tissue comprises TILs; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 to about 70 minutes; (e) freezing the vessel using the vapor phase of liquid nitrogen; (f) storing the vessel at the vapor phase of liquid nitrogen temperature; (g) thawing the tumor tissue; (h) adding the tumor tissue into a closed system; (i) performing in the closed system a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (i) to step (j) occurs without opening the system; (k) harvesting the therapeutic population of TILs obtained from step (j), wherein the transition from step (j) to step (k) occurs without opening the system; (1) transferring the therapeutic population of TILs harvested in step (k) to an infusion bag, wherein the transfer from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (1) using a cryopreservation process; (o) thawing the therapeutic population of TILs in the infusion bag from step (m); and (p) administering a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (o) to the subject.

[00386] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii)incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 30 minutes; (iv) freezing the vessel; and (v) storing the vessel such that the vessel remains frozen; (b) thawing the vessel; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (d) removing at least a plurality of the TILs; and (e) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00387] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 30 minutes; (e) freezing the vessel; (f) storing the vessel such that the vessel remains frozen; (g) thawing the vessel; (h) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (i) removing at least a plurality of the TILs; and (j) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00388] In some embodiments, the fresh tumor tissue is trimmed to between 1.5 mm and 6 mm in diameter and between about 1.5 mm and about 6 mm in thickness. In yet other embodiments, the fresh tumor tissue is trimmed to about 6 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 6 mm.

[00389] In some embodiments, the storage medium further comprises an antibacterial agent. In some embodiments the antibacterial agent is gentamicin. In some embodiments the storage medium comprises gentamicin at a concentration of at least 20 µg / mL. In some embodiments the storage medium comprises gentamicin at a concentration of at least 50 µg / mL. In some embodiments the storage medium comprises an antibacterial agent, an antifungal agent, and combinations thereof. In some embodiments, the antifungal agent is amphotericin B and is present from about 0.25 µg / mL to about 2.5 µg / mL. In some embodiments, the antifungal agent is fungin and is present from about 5 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>g / mL to about 50 <semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>g / mL.

[00390] In some embodiments, the fresh tumor tissue is first washed in Hank's Balanced Salt Solution (HBSS) before performing the trimming step of the method. In some embodiments, the washing comprises at least three serial washes of at least three minutes each, wherein the HBSS is replaced after each wash.

[00391] In some embodiments, thawing step comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[00392] In some embodiments, the freezing step comprises freezing the vessel at about -125°C to about -195°C. In some embodiments, the vessel is frozen at about -125°C to about -150°C; in some embodiments the vessel is frozen at about -125°C to about -145°C; in yet further embodiments, the vessel is frozen at about -135°C.

[00393] In some embodiments, the method is performed with a fresh tumor sample from a human subject.

[00394] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC), and HPV-positive tumor tissue.

[00395] In some embodiments, the method further comprises a split between the first expansion (or first culture) step and the second expansion (or second culture) step. In an embodiment, the split occurs on day 16. In some embodiments, the first culture step is complete in about 11 days. In other embodiments, the first expansion (or first culture) step is complete in about 7 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 7 days and the second expansion (or second culture) step is complete in about 14 days. In some embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are complete in about 22 days. In yet other embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are complete in 21 days; in some embodiments steps (c) through (e) or steps (h) through (j), as applicable, are complete in about 20 days; and in some embodiments steps (c) through (e) or steps (h) through (j), as applicable, are complete in about 16 days.

[00396] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof, such methods comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 20 to about 70 minutes; (iv) freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (b) thawing the tumor tissue; (c) adding the tumor fragments into a closed system; (d) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein 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 population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the therapeutic population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested TIL population from step (g) using a cryopreservation process; and (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject.

[00397] In some embodiments, the invention provides methods of treating cancer for a human subject in need thereof, such methods comprising: (a) obtaining tumor tissue from the subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 20 to about 70 minutes; (e) freezing the vessel using the vapor phase of liquid nitrogen; (f) storing the vessel at the vapor phase of liquid nitrogen temperature; (g) thawing the tumor tissue; (h) adding the tumor tissue into a closed system; (i) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (i) to step (j) occurs without opening the system; (k) harvesting the therapeutic population of TILs obtained from step (j), wherein the transition from step (j) to step (k) occurs without opening the system; (1) transferring the therapeutic population of TILs harvested in step (k) to an infusion bag, wherein the transfer from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (1) using a cryopreservation process; (n) thawing the therapeutic population of TILs in infusion bag from step (m); and (o) administering a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (n) to the subject.

[00398] In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC) glioblastoma, endometrial cancer, thyroid cancer, colorectal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, melanoma, refractory melanoma, metastatic melanoma, and non-small cell lung carcinoma. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment.

[00399] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising incubating the tumor tissue for about 20 to about 70 minutes in a storage medium at a temperature in the range from about 2°C to about 8°C before freezing; (c) thawing the tumor tissue; (d) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (e) removing at least a plurality of the TILs; and (f) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00400] The invention provides a method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) incubating the tumor tissue for about 20 to about 70 minutes in a storage medium at a temperature in the range from about 2°C to about 8°C; (c) storing the tumor tissue in a frozen state; (d) thawing the tumor tissue; (e) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (f) removing at least a plurality of the TILs; and (g) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00401] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 30 minutes; (iv) freezing the vessel; and (v) storing the vessel such that the vessel remains frozen; (c) thawing the vessel; (d) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (e) removing at least a plurality of the TILs; and (f) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs.

[00402] In some embodiments, the fresh tumor tissue is first washed in Hank's Balanced Salt Solution (HBSS) before performing the tissue trimming step. In some embodiments, the washing comprises at least three serial washes of at least three minutes each, wherein the HBSS is replaced after each wash.

[00403] In some embodiments the thawing step comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[00404] In some embodiments, the freezing step comprises freezing the vessel at a temperature between about -125°C to about -195°C. In some embodiments, the vessel is frozen at a temperature between about -125°C to about -150°C; in some embodiments the vessel is frozen at a temperature between about -125°C to about -145°C; in yet further embodiments, the vessel is frozen at a temperature of about -135°C.

[00405] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC), and HPV-positive tumor tissue.

[00406] In some embodiments, the method further comprises a split between the first expansion (or first culture) step and the second expansion (or second culture) step. In an embodiment, the split occurs on day 16. In some embodiments, the first culture step is complete in about 11 days. In other embodiments, the first expansion (or first culture) step is complete in about 7 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 7 days and the second expansion (or second culture) step is complete in about 14 days. In some embodiments, steps (d) through (f) or steps (e) through (g), as applicable, are complete in about 22 days. In yet other embodiments, steps (d) through (f) or steps (e) through (g), as applicable, are complete in about 21 days; in some embodiments steps (d) through (f) or steps (e) through (g), as applicable, are complete in about 20 days; and in some embodiments steps (d) through (f) or steps (e) through (g), as applicable, are complete in about 16 days.

[00407] In some embodiments, the invention provides for methods of treating cancer for a human subject in need thereof, such methods comprising: (a) obtaining tumor tissue from the subject, wherein the tumor tissue comprises TILs; (b) storing the tumor tissue in a frozen state, the method of storing the tumor tissue (1) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range from about 2°C to about 8°C for about 20 minutes to about 70 minutes; (iv) freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (c) thawing the tumor tissue; (d) adding the tumor tissue into a closed system; (e) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (e) to step (f) occurs without opening the system; (g) harvesting the therapeutic population of TILs obtained from step (f), wherein the transition from step (f) to step (g) occurs without opening the system; (h) transferring the harvested TIL population from step (g) to an infusion bag, wherein the transfer from step (g) to (h) occurs without opening the system; (i) cryopreserving the infusion bag comprising the harvested TIL population from step (h) using a cryopreservation process; and (i) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (i) to the subject.

[00408] In some embodiments, the fresh tumor tissue is first washed in Hank's Balanced Salt Solution (HBSS) before performing step (i) of the tumor tissue storing method. In some embodiments, the washing comprises at least three serial washes of at least three minutes each, wherein the HBSS is replaced after each wash.

[00409] In some embodiments step (c) comprises immersing the vessel in a 37°C water bath for about 5 minutes.

[00410] In some embodiments, step (iv) of the tumor tissue storing method comprises freezing the vessel at a temperature between about -125°C to about -195°C. In some embodiments, the vessel is frozen at a temperature between about -125°C to about -150°C; in some embodiments the vessel is frozen at a temperature between about -125°C to about -145°C; in yet further embodiments, the vessel is frozen at a temperature of about -135°C.

[00411] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, endometrial tumor tissue, thyroid tumor tissue, ovarian tumor tissue, head and neck squamous cell carcinoma (HNSCC), and HPV-positive tumor tissue.

[00412] In some embodiments, the method further comprises a split between the first expansion (or first culture) step and the second expansion (or second culture) step. In an embodiment, the split occurs on day 16. In some embodiments, the first culture step is complete in about 11 days. In other embodiments, the first expansion (or first culture) step is complete in about 7 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 7 days and the second expansion (or second culture) step is complete in about 14 days. In some embodiments, steps (e) through (g) are complete in about 22 days. In yet other embodiments, steps (e) through (g) are complete in about 21 days; in some embodiments steps (e) through (g) are complete in about 20 days; and in some embodiments steps (e) through (g) are complete in about 16 days.

[00413] In some embodiments, the first expansion (or first culture) step is complete in about 5 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 3 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 5 days and the second expansion (or second culture) step is complete in about 11 days. In yet other embodiments, the first expansion (or first culture) step is complete in about 3 days and the second expansion (or second culture) step is complete in about 13 days. Optionally, the second expansion (or second culture) can be split into two or more cultures on or about the fifth day or sixth day of the second expansion (or second culture).

[00414] In some embodiments of the above methods to manufacture or expand TILs, OKT-3 is present in the culture medium beginning at day 0.

[00415] In some embodiments, the invention provides any of the foregoing methods of treating cancer in a subject modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days.

[00416] In some embodiments, the invention provides any of the foregoing methods of treating cancer in a subject modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days.

[00417] In some embodiments, the invention provides any of the foregoing methods of treating cancer in a subject modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days and the second expansion (or second culture) is complete in about 11 days.

[00418] In some embodiments, the invention provides any of the foregoing methods of treating cancer in a subject modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days, the second expansion (or second culture) is complete in about 11 days, and the second expansion (or second culture) is split into two or more cultures on or about the fifth day of the second expansion (or second culture).

[00419] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days and the second expansion (or second culture) is complete in about 13 days.

[00420] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days, the second expansion (or second culture) is complete in about 13 days, and the second expansion (or second culture) is split into two or more cultures on or about the sixth day of the second expansion (or second culture).

[00421] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days.

[00422] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days.

[00423] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days and the second expansion (or second culture) is complete in about 11 days.

[00424] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days, the second expansion (or second culture) is complete in about 11 days, and the second expansion (or second culture) is split into two or more cultures on or about the fifth day of the second expansion (or second culture).

[00425] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days and the second expansion (or second culture) is complete in about 13 days.

[00426] In some embodiments, the invention provides any of the foregoing methods of expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days, the second expansion (or second culture) is complete in about 13 days, and the second expansion (or second culture) is split into two or more cultures on or about the sixth day of the second expansion (or second culture).

[00427] In some embodiments, the invention provides any of the foregoing methods for preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days and the second expansion (or second culture) is complete in about 13 days.

[00428] In some embodiments, the invention provides any of the foregoing methods of preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days, the second expansion (or second culture) is complete in about 13 days, and the second expansion (or second culture) is split into two or more cultures on or about the sixth day of the second expansion (or second culture).

[00429] In some embodiments, the invention provides any of the foregoing methods of preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days.

[00430] In some embodiments, the invention provides any of the foregoing methods of preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days.

[00431] In some embodiments, the invention provides any of the foregoing methods of preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days and the second expansion (or second culture) is complete in about 11 days.

[00432] In some embodiments, the invention provides any of the foregoing methods of preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 5 days, the second expansion (or second culture) is complete in about 11 days, and the second expansion (or second culture) is split into two or more cultures on or about the fifth day of the second expansion (or second culture).

[00433] In some embodiments, the invention provides any of the foregoing methods of preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days and the second expansion (or second culture) is complete in about 13 days.

[00434] In some embodiments, the invention provides any of the foregoing methods of preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy modified as appropriate such that in the step of the first expansion (or first culture) the first expansion (or first culture) is complete in about 3 days, the second expansion (or second culture) is complete in about 13 days, and the second expansion (or second culture) is split into two or more cultures on or about the sixth day of the second expansion (or second culture).

[00435] In some embodiments, the invention provides any of the foregoing methods modified as appropriate such that the first expansion (or first culture) is performed in a defined medium.

[00436] In some embodiments, the invention provides any of the foregoing methods modified as appropriate such that the second expansion (or second culture) is performed in a defind medium.

[00437] In some embodiments, the invention provides any of the foregoing methods modified as appropriate such that the first expansion (or first culture) and the second expansion (or second culture) are performed in defined media that are the same or different. TIL Manufacturing Processes

[00438] There are various methods to expand TILs known to one skilled in the art. For example, Jin et al., J. Immunother. 35(3): 283-292 (2012), "Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes in Gas-permeable Flasks to Numbers Needed for Patient Treatment," teaches simplified methods of producing TILs for clinical use. Jin et al. teaches a first TIL culture followed by a rapid expansion (REP) protocol, which combined, enables one skilled in the art to produce clinically useful quantities of TILs. In some embodiments, the invention provides a method of manufacturing TILs comprising the step of cryopreserving a tumor, thawing a tumor, and performing the process described in Jin et al. Briefly, this process involves the following process. TILs may initially be cultured from enzymatic tumor digests and tumor fragments (about 1 to 8 mm3) produced by sharp dissection. Tumor digests are produced by incubation in enzyme media (RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30U / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). Immediately after placing the tumor in enzyme media, it is mechanically dissociated for approximately 1 minute. The material was then incubated for 30 minutes at 37°C in 5% CO2 and is then mechanically disrupted again for approximately 1 minute and incubated again for 30 minutes at 37°C in 5% CO2. The tumor is then mechanically disrupted a third time for approximately 1 minute. If after the third mechanical disruption, large pieces of tissue were present, 1 or 2 additional mechanical dissociations may be applied to the sample, with or without 30 additional minutes of incubation at 37°C in 5% CO2. At the end of the final incubation, if the cell suspension contained a large number of red blood cells or dead cells, a density gradient separation using Ficoll may be performed to remove these cells. When TIL cultures are initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, NY), each well is seeded with <semantics>1×106<annotation encoding="application / x-tex">1 \times 10^6< / annotation>< / semantics> tumor digest cells or one tumor fragment approximately about 1 to 8 mm3 in size in 2 mL of complete medium (CM) with IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). CM comprised RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25mM Hepes, and about 10 μg / mL gentamicin. When cultures were initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2 gas-permeable silicon bottom (G- Rex10; Wilson Wolf Manufacturing, New Brighton, MN), each flask is loaded with 10 to <semantics>40×106<annotation encoding="application / x-tex">40 \times 10^6< / annotation>< / semantics> viable tumor digest cells or 5 to 30 tumor fragments in 10 to 40 mL of CM with IL-2. Both the G-Rex10 and 24-well plates are incubated in a humidified incubator at 37°C in 5%CO2 and 5 days after culture initiation, half the media is removed and replaced with fresh CM and IL-2 and after day 5, half the media is changed every 2-3 days. REP of TIL is performed using T-175 flasks and gas-permeable bags or gas-permeable G-Rex flasks. For TIL REP in T-175 flasks, 1×106 TILs suspended in 150 mL of media was added to each T-175 flask. The TIL were cultured with irradiated (50 Gy) allogeneic PBMC as "feeder" cells at a ratio of 1 to 100 and the cells are cultured in a 1 to 1 mixture of CM and AIM-V medium (50 / 50 medium), supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. The T-175 flasks are incubated at 37°C in 5% CO2. Half the media is changed on day 5 using 50 / 50 medium with 3000 IU / mL of IL-2. On day 7, cells from 2 T-175 flasks are combined in a 3L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU / mL of IL-2 is added to the 300 mL of TIL suspension. The number of cells in each bag was counted every day or 2 and fresh media was added to keep the cell count between 0.5 and 2.0×106 cells / mL. For TIL REP in 500mL capacity flasks with 100 cm2 gas-permeable silicon bottoms (G-Rex100, Wilson Wolf), 5×106 to 10×106 TIL are cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50 / 50 medium, supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. G-Rex100 flasks are incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant is removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. TIL pellets are resuspended with 150 mL of fresh 50 / 50 medium with 3000 IU / mL of IL-2 and added back to the original G-Rex100 flasks. When TIL are expanded serially in G-Rex100 flasks, on day 7 the TIL in each G-Rex100 were suspended in the 300 mL of media present in each flask and the cell suspension was divided into three 100 mL aliquots that were used to seed 3 G-Rex100 flasks. 150 mL of AIM-V with 5% human AB serum and 3000 IU / mL of IL-2 is added to each flask. The G-Rex100 flasks are incubated at 37°C in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU / mL of IL-2 was added to each G-Rex100 flask. The cells are harvested on day 14 of culture.

[00439] In some embodiments, the expansion methods according to the present invention comprises preparing expansion of cells from frozen tumor. Tumor samples are collected from patients and cryopreserved according to methods disclosed herein. When ready to expand the cells, for a process performed at the 1 / 100th scale, about six 2-3 mm diameter frozen tumor fragments are thawed using a <semantics>37∘<annotation encoding="application / x-tex">37^{\circ}< / annotation>< / semantics>C water bath for <semantics>5+1<annotation encoding="application / x-tex">5 + 1< / annotation>< / semantics> minutes and washed in sterile Hanks Balanced Salt Solution (HBSS) supplemented with gentamicin. The fragments are then placed into a G-Rex 10M flask (Wilson Wolf Mfg., New Brighton, MN) or other gas permeable container with CM1 media or a serum-free or defined media containing about 6,000 IU / mL of rhIL-2. These pre-REP (or first expansion) cultures are incubated for 5 days in a 37°C incubator. On day 5, the pre-REP cells are harvested, and the REP (or second expansion) is initiated by co-culturing 10% of the pre-REP TILs with either 25x106 or 50x106 PBMC feeder cells in CM2 media or a serum-free or defined media containing 3,000 IU / mL rhIL-2 and 30 ng / mL of OKT-3 in a G-Rex 5M flask (Wilson Wolf Mfg., New Brighton, MN) or other gas permeable container. On day 10, the culture is split into G-Rex 5M flasks or other gas permeable container containing no more than <semantics>10x106<annotation encoding="application / x-tex">10x10^6< / annotation>< / semantics> cells. The split cultures are incubated for 6 additional days in CM4 media or other serum-free or defined media containing 3,000 IU / mL rhIL-2. On day 16, the cells are harvested and frozen using CryoStor10 (Biolife, USA). This process is referred to herein as the "Early REP Method 1" process or "ER-1."

[00440] In some embodiments, the expansion methods according to the present invention comprises preparing expansion of cells from frozen tumor. Tumor samples are collected from patients and cryopreserved according to methods disclosed herein. When ready to expand the cells, for a process performed at the 1 / 100th scale, about six 2-3 mm diameter frozen tumor fragments are thawed using a <semantics>37∘<annotation encoding="application / x-tex">37^{\circ}< / annotation>< / semantics>C water bath for <semantics>5+1<annotation encoding="application / x-tex">5 + 1< / annotation>< / semantics> minutes and washed in sterile Hanks Balanced Salt Solution (HBSS) supplemented with gentamicin. The fragments are then placed into a G-Rex 10M flask (Wilson Wolf Mfg., New Brighton, MN) or other gas permeable container with CM1 media or a serum-free or defined media containing about 6,000 IU / mL of rhIL-2. These pre-REP (or first expansion) cultures are incubated for 3 days in a 37°C incubator. On day 3, the pre-REP cells are harvested, and the REP (or second expansion) is initiated by co- culturing 10% of the pre-REP TILs with either 25x106 or 50x106 PBMC feeder cells in CM2 media or a serum-free or defined media containing 3,000 IU / mL rhIL-2 and 30 ng / mL of OKT-3 in a G-Rex 5M flask (Wilson Wolf Mfg., New Brighton, MN) or other gas permeable container. On day 9, the culture is split into G-Rex 5M flasks or other gas permeable container containing no more than <semantics>10×106<annotation encoding="application / x-tex">10 \times 10^6< / annotation>< / semantics> cells. The split cultures are incubated for 7 additional days in CM4 media or other serum-free or defined media containing 3,000 IU / mL rhIL-2. On day 16, the cells are harvested and frozen using CryoStor10 (Biolife, USA). This process is referred to herein as the "Early REP Method 2" process or "ER-2."

[00441] In some embodiments, the invention provides a method of manufacturing TILs comprising the step of cryopreserving a tumor, thawing a tumor, and performing the following process. TILs can be produced by rapid expansion using stimulation of peripheral blood mononuclear cells (PBMC) in vitro with an antigen (one or more, including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as an HLA-A2 binding peptide, e.g., 0.3 μM MART-1:26-35 (27L) or gp100:209- 217 (210M), in the presence of a T-cell growth factor, such as 300 IU / mL IL-2 or IL-15, with IL- 2 being preferred. The in vitro-induced TILs are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example. TILs can be selected for highly avid recognition of any of the unique antigens produced as a result of the estimated 10,000 genetic mutations encoded by each tumor cell genome. The antigen, however, need not be unique. T-cells can be selected for highly avid recognition of one or more antigens of a cancer, including an antigenic portion of one or more antigens, such as an epitope, or a cell of the cancer. An "antigen of a cancer" and an "antigen of the cancer" are intended to encompass all of the aforementioned antigens. If the cancer is melanoma, such as metastatic melanoma, preferably the TILs are selected for highly avid recognition of MART-1 (such as MART-1:26- 35 (27L)), gp100 (such as gp100:209-217 (210M)), or a "unique" or patient-specific antigen derived from a tumor encoded mutation. Other suitable melanoma antigens for which highly avid recognition by TILs can be selected include, but are not limited to, tyrosinase, tyrosinase related protein (TRP)1, TRP2, and MAGE. Antigens, such as NY-ESO-1, telomerase, p53, HER2 / neu, carcinoembryonic antigen, or prostate-specific antigen, can be used to select for highly avid recognition by TILs for treatment of lung carcinoma, breast cancer, colon cancer, prostate cancer, and the like TILs can be selected include, but are not limited to, tyrosinase, tyrosinase related protein.

[00442] IL-2-based TIL expansion followed by a "rapid expansion process" (REP) has become a preferred method for TIL expansion because of its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-fold expansion of TILs over a 14-day period, although it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs), often from multiple donors, as feeder cells, as well as anti- CD3 antibody (OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. The cryopreserved tumor fragments are suitable starting points for initial or first cultures to manufacture TILs for therapeutic or other purposes.

[00443] An exemplary TIL process known as process 2A containing some of these features is depicted in Figure 1. Another exemplary TIL process, known as process 1C, is described and compared to process 2A in Figures 5 and 6. An embodiment of process 2A is shown Figure 1.

[00444] As discussed herein, the present invention can include a step relating to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplant into a patient, and methods of testing said metabolic health. As generally outlined herein, TILs are generally taken from a patient sample and manipulated to expand their number prior to transplant into a patient. In some embodiments, the TILs may be optionally genetically manipulated as discussed below.

[00445] In some embodiments, the TILs may be cryopreserved and thawed for administration to a patient. Once thawed, they may also be restimulated to increase their metabolism prior to infusion into a patient.

[00446] In some embodiments, the first expansion (including processes referred to as the preREP as well as processes shown in Figure 1 as Step A) is shortened to 3 to 14 days and the second expansion (including processes referred to as the REP as well as processes shown in Figure 1 as Step B) is shorted to 7 to 14 days, as discussed in detail below as well as in the examples and figures. In some embodiments, the first expansion (for example, an expansion described as Step B in Figure 1) is shortened to 11 days and the second expansion (for example, an expansion as described in Step D in Figure 1) is shortened to 11 days. In some embodiments, the combination of the first expansion and second expansion (for example, expansions described as Step B and Step D in Figure 1) is shortened to 22 days, as discussed in detail below and in the examples and figures.

[00447] The "Step" Designations A, B, C, etc., below are in reference to Figure 1 and in reference to certain embodiments described herein. The ordering of the Steps below and in Figure 1 is exemplary and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps is contemplated by the present application and the methods disclosed herein. Step A. Obtain Patient Tumor Sample

[00448] In general, TILs are initially obtained from a patient tumor sample ("primary TILs") and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health.

[00449] A patient tumor sample may be obtained using methods known in the art, generally via surgical resection, needle biopsy or other means for obtaining a sample that contains a mixture of tumor and TIL cells. In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, renal, stomach, and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these have been reported to have particularly high levels of TILs.

[00450] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. The term "solid tumor cancer" refers to malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, triple negative breast cancer, prostate, colon, rectum, and bladder. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC) glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small cell lung carcinoma. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment.

[00451] The term "hematological malignancy" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term "B cell hematological malignancy" refers to hematological malignancies that affect B cells.

[00452] Once obtained, the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm3, with from about 2-3 mm3 being particularly useful. The TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests may be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL of DNase and 1.0 mg / mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 A1. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.

[00453] In general, the harvested cell suspension is called a "primary cell population" or a "freshly harvested" cell population.

[00454] In some embodiments, fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is dissection. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients. In an embodiment, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.

[00455] In some embodiments, where the tumor is a solid tumor, the tumor undergoes physical fragmentation after the tumor sample is obtained in, for example, Step A (as provided in Figure 1). In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after obtaining the tumor and in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm3. In some embodiments, the multiple fragments comprise about 30 to about 60 fragments with a total volume of about 1300 mm3 to about 1500 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total volume of about 1350 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments comprise about 4 fragments.

[00456] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragment is obtained by sharp dissection. In some embodiments, the tumor fragment is between about 1 mm3 and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3 and 8 mm3. In some embodiments, the tumor fragment is about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragment is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment is about 10 mm3. In some embodiments, the tumors are 1-4 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1-4 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1-4 mm. In some embodiments, the tumors are 1 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 1 mm. In some embodiments, the tumors are 2 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 2 mm. In some embodiments, the tumors are 3 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 3 mm. In some embodiments, the tumors are 4 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4 mm <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 4 mm. In embodiments wherein the tumor tissue is first frozen before initiating a culture, tumors are about <semantics>6 mm×6 mm×6 mm<annotation encoding="application / x-tex">6 \text{ mm} \times 6 \text{ mm} \times 6 \text{ mm}< / annotation>< / semantics>.

[00457] In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic, necrotic, and / or fatty tissues on each piece. In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of fatty tissue on each piece.

[00458] In some embodiments, the tumor fragmentation is performed in order to maintain the tumor internal structure. In some embodiments, the tumor fragmentation is performed without preforming a sawing motion with a scalpel. In some embodiments, the TILs are obtained from tumor digests. In some embodiments, tumor digests were generated by incubation in enzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 μg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37 °C in 5% CO2 and it then mechanically disrupted again for approximately 1 minute. After being incubated again for 30 minutes at 37 °C in 5% CO2, the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37 °C in 5% CO2. In some embodiments, at the end of the final incubation if the cell suspension contained a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

[00459] In some embodiments, the harvested cell suspension prior to the first expansion step is called a "primary cell population" or a "freshly harvested" cell population.

[00460] In some embodiments, cells can be optionally frozen after sample harvest and stored frozen prior to entry into the expansion described in Step B, which is described in further detail below, as well as exemplified in Figure 1. Step B: First Expansion

[00461] In some embodiments, the present methods provide for obtaining young TILs, which are capable of increased replication cycles upon administration to a subject / patient and as such may provide additional therapeutic benefits over older TILs (for example "older TILs" have further undergone more rounds of ex vivo replication prior to administration to a subject / patient). Features of young TILs have been described in the literature, for example Donia, et al., Scandinavian Journal of Immunology, 75:157–167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258–267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother, 32:415–423 (2009); Robbins, et al., J Immunol, 2004; 173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, et al., J Immunother, 28:53–62 (2005); and Tran, et al., J Immunother, 31:742–751 (2008).

[00462] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited, but large number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs). The present invention provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and / or TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and / or TILs prepared using methods referred to as process 1C, as exemplified in Figure 5 and / or Figure 6. In some embodiments, the TILs obtained in the first expansion exhibit an increase in the T-cell repertoire diversity. In some embodiments, the increase in diversity is an increase in the immunoglobulin diversity and / or the T-cell receptor diversity. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. In some embodiments, the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha and / or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCR<semantics>α / β<annotation encoding="application / x-tex">\alpha / \beta< / annotation>< / semantics>).

[00463] After dissection or digestion of tumor fragments, for example such as described in Step A of Figure 1, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 3 to 14 days, resulting in a bulk TIL population, generally about <semantics>1×108<annotation encoding="application / x-tex">1 \times 10^8< / annotation>< / semantics> bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, resulting in a bulk TIL population, generally about <semantics>1×108<annotation encoding="application / x-tex">1 \times 10^8< / annotation>< / semantics> bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 10 to 14 days, resulting in a bulk TIL population, generally about <semantics>1×108<annotation encoding="application / x-tex">1 \times 10^8< / annotation>< / semantics> bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about <semantics>1×108<annotation encoding="application / x-tex">1 \times 10^8< / annotation>< / semantics> bulk TIL cells.

[00464] In a preferred embodiment, expansion of TILs may be performed using an initial bulk TIL expansion step (for example such as those described in Step B of Figure 1, which can include processes referred to as pre-REP) as described below and herein, followed by a second expansion (Step D, including processes referred to as rapid expansion protocol (REP) steps) as described below under Step D and herein, followed by optional cryopreservation, and followed by a second Step D (including processes referred to as restimulation REP steps) as described below and herein. The TILs obtained from this process may be optionally characterized for phenotypic characteristics and metabolic parameters as described herein.

[00465] In embodiments where TIL cultures are initiated in 24-well plates, for example, using Costar 24-well cell culture cluster, flat bottom (Corning Incorporated, Corning, NY, each well can be seeded with <semantics>1×106<annotation encoding="application / x-tex">1 \times 10^6< / annotation>< / semantics> tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is between about 1 mm3 and 10 mm3.

[00466] In some embodiments, the first expansion culture medium is referred to as "CM", an abbreviation for culture media. In some embodiments, CM for Step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 μg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2 gas-permeable silicon bottom (for example, G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Fig. 1), each flask was loaded with <semantics>10×106<annotation encoding="application / x-tex">10 \times 10^6< / annotation>< / semantics> to <semantics>40×106<annotation encoding="application / x-tex">40 \times 10^6< / annotation>< / semantics> viable tumor digest cells or 5 to 30 tumor fragments in 10 to 40 mL of CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5, half the media was changed every 2 to 3 days.

[00467]

[00468] After preparation of the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of aAPC cell population) with 6000 IU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL population, generally about <semantics>1×108<annotation encoding="application / x-tex">1\times10^8< / annotation>< / semantics> bulk TIL cells. In some embodiments, the growth media during the first expansion comprises IL- 2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30×106 IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20×106 IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25×106 IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of <semantics>30×106<annotation encoding="application / x-tex">30 \times 10^6< / annotation>< / semantics> IU / mg for a 1 mg vial. In some embodiments, the IL- 2 stock solution has a final concentration of 4-8×106 IU / mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7×106 IU / mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of <semantics>6×106<annotation encoding="application / x-tex">6\times10^6< / annotation>< / semantics> IU / mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in Example E. In some embodiments, the first expansion culture media comprises about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6000 IU / mL of IL-2 or about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 9,000 IU / mL of IL-2 to about 5,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 8,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 6,000 IU / mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In an embodiment, the cell culture medium comprises between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, or about 8000 IU / mL of IL-2.

[00469] In some embodiments, first expansion culture media comprises about 500 IU / mL of IL- 15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL- 15, or about 100 IU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture media comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In an embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[00470] In some embodiments, first expansion culture media comprises about 20 IU / mL of IL- 21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture media comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In an embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[00471] In an embodiment, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 µg / mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[00472] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4- 1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 µg / mL and 100 µg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 µg / mL and 40 µg / mL.

[00473] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.

[00474] In some embodiments, the first expansion culture medium is referred to as "CM", an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeable flasks with a 40 mL capacity and a 10cm2 gas-permeable silicon bottom (for example, G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Fig. 1), each flask was loaded with <semantics>10×106<annotation encoding="application / x-tex">10 \times 10^6< / annotation>< / semantics> to <semantics>40×106<annotation encoding="application / x-tex">40 \times 10^6< / annotation>< / semantics> viable tumor digest cells or 5–30 tumor fragments in 10 to 40mL of CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5, half the media was changed every 2 to 3 days. In some embodiments, the CM is the CM1 described in the Examples, see, Example 1. In some embodiments, the first expansion occurs in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium comprises IL-2.

[00475] In some embodiments, the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre- REP) process is shortened to 3 to 14 days, as discussed in the examples and figures. In some embodiments, the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre-REP) is shortened to 7 to 14 days, as discussed in the Examples and shown in Figures 4 and 5, as well as including for example, an expansion as described in Step B of Figure 1. In some embodiments, the first expansion of Step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, as discussed in, for example, an expansion as described in Step B of Figure 1.

[00476] In some embodiments, the first TIL expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 14 days. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 4 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 7 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 9 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 11 days to 14 days. In some embodiments, the first TIL expansion can proceed for 12 days to 14 days. In some embodiments, the first TIL expansion can proceed for 13 days to 14 days. In some embodiments, the first TIL expansion can proceed for 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 11 days. In some embodiments, the first TIL expansion can proceed for 2 days to 11 days. In some embodiments, the first TIL expansion can proceed for 3 days to 11 days. In some embodiments, the first TIL expansion can proceed for 4 days to 11 days. In some embodiments, the first TIL expansion can proceed for 5 days to 11 days. In some embodiments, the first TIL expansion can proceed for 6 days to 11 days. In some embodiments, the first TIL expansion can proceed for 7 days to 11 days. In some embodiments, the first TIL expansion can proceed for 8 days to 11 days. In some embodiments, the first TIL expansion can proceed for 9 days to 11 days. In some embodiments, the first TIL expansion can proceed for 10 days to 11 days. In some embodiments, the first TIL expansion can proceed for 11 days.

[00477] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 are employed as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL- 21 as well as any combinations thereof can be included during the first expansion, including for example during a Step B processes according to Figure 1, as well as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the first expansion. In some embodiments, IL-2, IL-15, and IL-21 as well as any combinations thereof can be included during Step B processes according to Figure 1 and as described herein.

[00478] In some embodiments, the first expansion (including processes referred to as the pre- REP; for example, Step B according to Figure 1) process is shortened to 3 to 14 days, as discussed in the examples and figures. In some embodiments, the first expansion of Step B is shortened to 7 to 14 days. In some embodiments, the first expansion of Step B is shortened to 10 to 14 days. In some embodiments, the first expansion is shortened to 11 days.

[00479] In some embodiments, the first expansion, for example, Step B according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX - 100. In some embodiments, the closed system bioreactor is a single bioreactor. Step C: First Expansion to Second Expansion Transition

[00480] In some cases, the bulk TIL population obtained from the first expansion, including for example the TIL population obtained from for example, Step B as indicated in Figure 1, can be cryopreserved immediately, using the protocols discussed herein below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, can be subjected to a second expansion (which can include expansions sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, in the case where genetically modified TILs will be used in therapy, the first TIL population (sometimes referred to as the bulk TIL population) or the second TIL population (which can in some embodiments include populations referred to as the REP TIL populations) can be subjected to genetic modifications for suitable treatments prior to expansion or after the first expansion and prior to the second expansion.

[00481] In some embodiments, the TILs obtained from the first expansion (for example, from Step B as indicated in Figure 1) are stored until phenotyped for selection. In some embodiments, the TILs obtained from the first expansion (for example, from Step B as indicated in Figure 1) are not stored and proceed directly to the second expansion. In some embodiments, the TILs obtained from the first expansion are not cryopreserved after the first expansion and prior to the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs at about 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 3 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 10 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 14 days from when fragmentation occurs.

[00482] In some embodiments, the transition from the first expansion to the second expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 14 days from when fragmentation occurs. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days from when fragmentation occurs.

[00483] In some embodiments, the TILs are not stored after the first expansion and prior to the second expansion, and the TILs proceed directly to the second expansion (for example, in some embodiments, there is no storage during the transition from Step B to Step D as shown in Figure 1). In some embodiments, the transition occurs in closed system, as described herein. In some embodiments, the TILs from the first expansion, the second population of TILs, proceeds directly into the second expansion with no transition period.

[00484] In some embodiments, the transition from the first expansion to the second expansion, for example, Step C according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX-10 or a G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor. Cytokines

[00485] The expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.

[00486] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is additionally possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is generally outlined in International Publication No. WO 2015 / 189356 and W International Publication No. WO 2015 / 189357. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL- 21 and IL-2, IL-15 and IL-21, with the latter finding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein. Step D: Second Expansion

[00487] In some embodiments, the TIL cell population is expanded in number after harvest and initial bulk processing for example, after Step A and Step B, and the transition referred to as Step C, as indicated in Figure 1). This further expansion is referred to herein as the second expansion, which can include expansion processes generally referred to in the art as a rapid expansion process (REP; as well as processes as indicated in Step D of Figure 1). The second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells, a cytokine source, and an anti-CD3 antibody, in a gas-permeable container.

[00488] In some embodiments, the second expansion or second TIL expansion (which can include expansions sometimes referred to as REP; as well as processes as indicated in Step D of Figure 1) of TIL can be performed using any TIL flasks or containers known by those of skill in the art. In some embodiments, the second TIL expansion can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion can proceed for about 7 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 8 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 9 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 10 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

[00489] In an embodiment, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including for example, expansions referred to as REP; as well as processes as indicated in Step D of Figure 1). For example, TILs can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T-cell receptor stimulus can include, for example, an anti-CD3 antibody, such as about 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further stimulation of the TILs in vitro by including one or more antigens during the second expansion, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 µM MART-1:26-35 (27) L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further re- stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the re-stimulation occurs as part of the second expansion. In some embodiments, the second expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL- 2.

[00490] In an embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In an embodiment, the cell culture medium comprises between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, or between 8000 IU / mL of IL-2.

[00491] In an embodiment, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[00492] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4- 1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 µg / mL and 100 µg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 µg / mL and 40 µg / mL.

[00493] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.

[00494] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21 as well as any combinations thereof can be included during the second expansion, including for example during a Step D processes according to Figure 1, as well as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21 as well as any combinations thereof can be included during Step D processes according to Figure 1 and as described herein.

[00495] In some embodiments, the second expansion can be conducted in a supplemented cell culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen- presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen presenting cells).

[00496] In some embodiments, the second expansion culture media comprises about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture media comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In an embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[00497] In some embodiments, the second expansion culture media comprises about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the second expansion culture media comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In an embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[00498] In some embodiments the antigen-presenting feeder cells (APCs) are PBMCs. In an embodiment, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion and / or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 50 and 1 to 300. In an embodiment, the ratio of TILs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[00499] In an embodiment, REP and / or the second expansion is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 IU / mL IL-2 in 150 ml media. Media replacement is done (generally 2 / 3 media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.

[00500] In some embodiments, the second expansion (which can include processes referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second expansion is shortened to 11 days.

[00501] In an embodiment, REP and / or the second expansion may be performed using T-175 flasks and gas permeable bags as previously described (Tran, et al., J. Immunother, 2008, 31, 742-51; Dudley, et al., J. Immunother, 2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks). In some embodiments, the second expansion (including expansions referred to as rapid expansions) is performed in T-175 flasks, and about <semantics>1×106<annotation encoding="application / x-tex">1 \times 10^6< / annotation>< / semantics> TILs suspended in 150 mL of media may be added to each T-175 flask. The TILs may be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175 flasks may be incubated at 37° C in 5% CO2. Half the media may be exchanged on day 5 using 50 / 50 medium with 3000 IU per mL of IL-2. In some embodiments, on day 7 cells from two T-175 flasks may be combined in a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TIL suspension. The number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and <semantics>2.0×106<annotation encoding="application / x-tex">2.0 \times 10^6< / annotation>< / semantics> cells / mL.

[00502] In an embodiment, the second expansion (which can include expansions referred to as REP, as well as those referred to in Step D of Figure 1) may be performed in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), <semantics>5×106<annotation encoding="application / x-tex">5 \times 10^6< / annotation>< / semantics> or <semantics>10×106<annotation encoding="application / x-tex">10 \times 10^6< / annotation>< / semantics> TIL may be cultured with PBMCs in 400 mL of 50 / 50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100 flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL- 2 may be added to each G-REX 100 flask. The cells may be harvested on day 14 of culture.

[00503] In an embodiment, the second expansion (including expansions referred to as REP) is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 IU / mL IL-2 in 150 ml media. In some embodiments, media replacement is done until the cells are transferred to an alternative growth chamber. In some embodiments, 2 / 3 of the media is replaced by respiration with fresh media. In some embodiments, alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.

[00504] In an embodiment, the second expansion (including expansions referred to as REP) is performed and further comprises a step wherein TILs are selected for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. Patent Application Publication No. 2016 / 0010058 A1, may be used for selection of TILs for superior tumor reactivity.

[00505] Optionally, a cell viability assay can be performed after the second expansion (including expansions referred to as the REP expansion), using standard assays known in the art. For example, a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelor Bioscience, Lawrence, MA). In some embodiments, viability is determined according to the standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.

[00506] In some embodiments, the second expansion (including expansions referred to as REP) of TIL can be performed using T-175 flasks and gas-permeable bags as previously described (Tran KQ, Zhou J, Durflinger KH, et al., 2008, J Immunother., 31:742–751, and Dudley ME, Wunderlich JR, Shelton TE, et al. 2003, J Immunother., 26:332–342) or gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed in T-175 flasks, and about <semantics>1×106<annotation encoding="application / x-tex">1 \times 10^6< / annotation>< / semantics> TIL are suspended in about 150 mL of media and this is added to each T-175 flask. The TIL are cultured with irradiated (50 Gy) allogeneic PBMC as "feeder" cells at a ratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium (50 / 50 medium), supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. The T-175 flasks are incubated at 37°C in 5% CO2. In some embodiments, half the media is changed on day 5 using 50 / 50 medium with 3000 IU / mL of IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU / mL of IL-2 is added to the 300 mL of TIL suspension. The number of cells in each bag can be counted every day or two and fresh media can be added to keep the cell count between about 0.5 and about <semantics>2.0×106<annotation encoding="application / x-tex">2.0 \times 10^6< / annotation>< / semantics> cells / mL.

[00507] In some embodiments, the second expansion (including expansions referred to as REP) are performed in 500 mL capacity flasks with 100 cm2 gas-permeable silicon bottoms (G-Rex 100, Wilson Wolf) (Fig. 1), about <semantics>5×106<annotation encoding="application / x-tex">5 \times 10^6< / annotation>< / semantics> or <semantics>10×106<annotation encoding="application / x-tex">10 \times 10^6< / annotation>< / semantics> TIL are cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50 / 50 medium, supplemented with 3000 IU / mL of IL- 2 and 30 ng / mL of anti-CD3. The G-Rex 100 flasks are incubated at 37°C in 5% CO2. In some embodiments, on day 5, 250mL of supernatant is removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491g) for 10 minutes. The TIL pellets can then be resuspended with 150 mL of fresh 50 / 50 medium with 3000 IU / mL of IL-2 and added back to the original G-Rex 100 flasks. In embodiments where TILs are expanded serially in G-Rex 100 flasks, on day 7 the TIL in each G-Rex 100 are suspended in the 300 mL of media present in each flask and the cell suspension was divided into three 100 mL aliquots that are used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU / mL of IL-2 is added to each flask. The G-Rex 100 flasks are incubated at 37°C in 5% CO2 and after 4 days 150 mL of AIM- V with 3000 IU / mL of IL-2 is added to each G-Rex 100 flask. The cells are harvested on day 14 of culture.

[00508] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited, but large number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs). The present invention provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity. In some embodiments, the TILs obtained in the second expansion exhibit an increase in the T-cell repertoire diversity. In some embodiments, the increase in diversity is an increase in the immunoglobulin diversity and / or the T-cell receptor diversity. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. In some embodiments, the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, there is an increase in the expression of T- cell receptor (TCR) alpha and / or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCR<semantics>α / β<annotation encoding="application / x-tex">\alpha / \beta< / annotation>< / semantics>).

[00509] In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the antigen- presenting feeder cells (APCs), as discussed in more detail below.

[00510] In some embodiments, the second expansion, for example, Step D according to Figure 1, is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor. Feeder Cells and Antigen Presenting Cells

[00511] In an embodiment, the second expansion procedures described herein (for example including expansion such as those described in Step D from Figure 1, as well as those referred to as REP) require an excess of feeder cells during REP TIL expansion and / or during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation, see, e.g. "Isolation of mononuclear cells: Methodology and Application", GE Life Sciences technical publication 18- 1152-69-AE, available at https: / / us.vwr.com / assetsvc / asset / en US / id / 16286835 / contents.

[00512] In general, the allogenic PBMCs are inactivated, either via irradiation or heat treatment, and used in the REP procedures, as described in the examples, which provides an exemplary protocol for evaluating the replication incompetence of irradiate allogeneic PBMCs.

[00513] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion).

[00514] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 3000 IU / ml IL-2.

[00515] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 5 to 60 ng / ml OKT3 antibody and 1000-6000 IU / ml IL-2. In some embodiments, the PBMCs are cultured in the presence of 10 to 50 ng / ml OKT3 antibody and 2000-5000 IU / ml IL- 2. In some embodiments, the PBMCs are cultured in the presence of 20 to 40 ng / ml OKT3 antibody and 2000-4000 IU / ml IL-2. In some embodiments, the PBMCs are cultured in the presence of 25 to 35 ng / ml OKT3 antibody and 2500-3500 IU / ml IL-2.

[00516] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.

[00517] In an embodiment, the second expansion procedures described herein require a ratio of about <semantics>2.5×109<annotation encoding="application / x-tex">2.5 \times 10^9< / annotation>< / semantics> feeder cells to about <semantics>100×106<annotation encoding="application / x-tex">100 \times 10^6< / annotation>< / semantics> TILs. In another embodiment, the second expansion procedures described herein require a ratio of about <semantics>2.5×109<annotation encoding="application / x-tex">2.5 \times 10^9< / annotation>< / semantics> feeder cells to about 50 <semantics>×<annotation encoding="application / x-tex">\times< / annotation>< / semantics> 106 TILs. In yet another embodiment, the second expansion procedures described herein require about <semantics>2.5×109<annotation encoding="application / x-tex">2.5 \times 10^9< / annotation>< / semantics> feeder cells to about <semantics>25×106<annotation encoding="application / x-tex">25 \times 10^6< / annotation>< / semantics> TILs.

[00518] In an embodiment, the second expansion procedures described herein require an excess of feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In an embodiment, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[00519] In general, the allogenic PBMCs are inactivated, either via irradiation or heat treatment, and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.

[00520] In an embodiment, artificial antigen presenting cells are used in the second expansion as a replacement for, or in combination with, PBMCs. Cytokines

[00521] The TIL expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.

[00522] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is additionally possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is generally outlined in International Publication No. WO 2015 / 189356 and W International Publication No. WO 2015 / 189357. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL- 21 and IL-2, IL-15 and IL-21, with the latter finding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein. Step E: Harvest TILs

[00523] After the second expansion step, cells can be harvested. In some embodiments the TILs are harvested after one, two, three, four or more expansion steps, for example as provided in Figure 1. In some embodiments the TILs are harvested after two expansion steps, for example as provided in Figure 1.

[00524] TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such know methods can be employed with the present process. In some embodiments, TILS are harvest using an automated system.

[00525] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell based harvester can be employed with the present methods. In some embodiments, the cell harvester and / or cell processing systems is a membrane-based cell harvester. In some embodiments, cell harvesting is via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any vendor that can pump a solution comprising cells through a membrane or filter such as a spinning membrane or spinning filter in a sterile and / or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without pelletization. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid-exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[00526] In some embodiments, the harvest, for example, Step E according to Figure 1, is performed from a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX -100. In some embodiments, the closed system bioreactor is a single bioreactor.

[00527] In some embodiments, Step E according to Figure 1, is performed according to the processes described in Example 7. In some embodiments, the closed system is accessed via syringes under sterile conditions in order to maintain the sterility and closed nature of the system. In some embodiments, a closed system, as described in Example 7, is employed.

[00528] In some embodiments, TILs are harvested according to the methods described in Example 7. In some embodiments, TILs between days 1 and 11 are harvested using the methods as described herein (referred to as the Day 11 TIL harvest in Example 7). In some embodiments, TILs between days 12 and 22 are harvested using the methods as described herein (referred to as the Day 22 TIL harvest in Example 7). Step F: Final Formulation / Transfer to Infusion Bag

[00529] After Steps A through E as provided in an exemplary order in Figure 1 and as outlined in detailed above and herein are complete, cells are transferred to a container for use in administration to a patient. In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, they are transferred to a container for use in administrati...

Claims

<pat:ClaimStatement>CLAIMS We claim:< / pat:ClaimStatement> <pat:Claims com:id="claims"> <pat:Claim com:id="CLM-00001"> <pat:ClaimNumber>1< / pat:ClaimNumber> <pat:ClaimText>1. A method of cryopreserving tumor tissue for the manufacture of tumor infiltrating lymphocytes (TILs), the method comprising: (i) fragmenting the tumor tissue; (ii) incubating the fragments in a cryopreservation medium for about 30 minutes to about 80 minutes at a temperature of about 2° to 8°C; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00002"> <pat:ClaimNumber>2< / pat:ClaimNumber> <pat:ClaimText>2. The method of claim 1, wherein the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 6 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00003"> <pat:ClaimNumber>3< / pat:ClaimNumber> <pat:ClaimText>3. The method of claim 2, wherein the approximately spherical fragments have a diameter of about 6 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00004"> <pat:ClaimNumber>4< / pat:ClaimNumber> <pat:ClaimText>4. The method of claim 2, wherein the approximately spherical fragments have a diameter of about 3 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00005"> <pat:ClaimNumber>5< / pat:ClaimNumber> <pat:ClaimText>5. The method of claim 1, wherein the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 1.5 mm and a longest edge length of about 6 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00006"> <pat:ClaimNumber>6< / pat:ClaimNumber> <pat:ClaimText>6. The method of claim 1, wherein the tumor tissue is fragmented into generally cubical fragments having edge lengths of between about 1.5 mm and about 6 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00007"> <pat:ClaimNumber>7< / pat:ClaimNumber> <pat:ClaimText>7. The method of claim 6, wherein the generally cubical fragments have edge lengths of about 6 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00008"> <pat:ClaimNumber>8< / pat:ClaimNumber> <pat:ClaimText>8. The method of claim 6, wherein the generally cubical fragments have edge lengths of about 3 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00009"> <pat:ClaimNumber>9< / pat:ClaimNumber> <pat:ClaimText>9. The method of any one of claims 1-8, wherein the tumor tissue is from a dissected tumor. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00010"> <pat:ClaimNumber>10< / pat:ClaimNumber> <pat:ClaimText>10. The method of any one of claims 1-9, wherein the dissected tumor is less than eight hours old. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00011"> <pat:ClaimNumber>11< / pat:ClaimNumber> <pat:ClaimText>11. The method of any one of claims 1-10, wherein the cryopreservation medium comprises 2% v / v to 15% v / v dimethyl sulfoxide (DMSO). < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00012"> <pat:ClaimNumber>12< / pat:ClaimNumber> <pat:ClaimText>12. The method of claim 11, wherein the cryopreservation medium comprises about 10% v / v DMSO. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00013"> <pat:ClaimNumber>13< / pat:ClaimNumber> <pat:ClaimText>13. The method of any one of claims 1-12, wherein the cryopreservation medium comprises at least one antimicrobial agent. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00014"> <pat:ClaimNumber>14< / pat:ClaimNumber> <pat:ClaimText>14. The method of claim 13, wherein the at least one antimicrobial agent is gentamicin at a concentration of at least 50 µg / mL. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00015"> <pat:ClaimNumber>15< / pat:ClaimNumber> <pat:ClaimText>15. The method of any one of claims 1-14, wherein the tumor tissue is washed in a physiologically buffered isotonic saline solution prior to incubation. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00016"> <pat:ClaimNumber>16< / pat:ClaimNumber> <pat:ClaimText>16. The method of claim 15, wherein the washing comprises three serial washes of at least three minutes each, with the physiologically buffered isotonic saline solution replaced after each serial wash. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00017"> <pat:ClaimNumber>17< / pat:ClaimNumber> <pat:ClaimText>17. The method of any one of claims 1-16, wherein the freezing takes place at a temperature in the range from of -125°C to about -196°C. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00018"> <pat:ClaimNumber>18< / pat:ClaimNumber> <pat:ClaimText>18. The method of any one of claims 1-17, wherein the freezing takes place at a temperature of about -140°C to about -175°C. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00019"> <pat:ClaimNumber>19< / pat:ClaimNumber> <pat:ClaimText>19. The method of any one of claims 1-18, wherein the freezing takes place at a temperature of about -145°C. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00020"> <pat:ClaimNumber>20< / pat:ClaimNumber> <pat:ClaimText>20. The method of any one of claims 1-19, further comprising the step of (iv) storing the frozen fragments below at least -130°C. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00021"> <pat:ClaimNumber>21< / pat:ClaimNumber> <pat:ClaimText>21. The method of claim 20, wherein the frozen fragments are stored in the vapor phase of liquid nitrogen. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00022"> <pat:ClaimNumber>22< / pat:ClaimNumber> <pat:ClaimText>22. The method of claim 20, wherein the frozen fragments are stored submerged in liquid nitrogen. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00023"> <pat:ClaimNumber>23< / pat:ClaimNumber> <pat:ClaimText>23. A cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by a process comprising the steps of: (i) fragmenting a dissected tumor or tumor biopsy sample; (ii) incubating the fragments in a cryopreservation medium; and, (iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen; wherein step (ii) further comprises incubating the fragments for about 30 minutes to about 60 minutes at about 2°C to about 8°C in a cryopreservation medium comprising 10% v / v DMSO. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00024"> <pat:ClaimNumber>24< / pat:ClaimNumber> <pat:ClaimText>24. A method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy, the method comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature of about 2 to about 8°C for a period of about 30 minutes to about 60 minutes; (iv) freezing the vessel, wherein the freezing is flash freezing using the vapor phase of liquid nitrogen; and (v) storing the vessel at or below -130°C; (b) thawing the vessel; (c) treating the tumor tissue in a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) to provide TILs; (d) expanding the TILs in a gas permeable container using a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3, and IL-2, to provide an expanded number of TILs. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00025"> <pat:ClaimNumber>25< / pat:ClaimNumber> <pat:ClaimText>25. The method of claim 24, wherein step (c) further comprises OKT-3. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00026"> <pat:ClaimNumber>26< / pat:ClaimNumber> <pat:ClaimText>26. The method of claim 24 or 25, wherein the tumor tissue in step (i) is trimmed to between < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00027"> <pat:ClaimNumber>27< / pat:ClaimNumber> <pat:ClaimText>27. The method of any one of claims 24-26, wherein the tumor tissue in step (i) is trimmed to about 6 mm by 6 mm by 6 mm. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00028"> <pat:ClaimNumber>28< / pat:ClaimNumber> <pat:ClaimText>28. The method of any one of claims 24 to 27, wherein the storage medium further comprises an antibacterial agent and an antifungal agent. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00029"> <pat:ClaimNumber>29< / pat:ClaimNumber> <pat:ClaimText>29. The method of any one of claims 24 to 28, wherein the storage medium comprises 2 % v / v to 12% v / v dimethyl sulfoxide (DMSO). < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00030"> <pat:ClaimNumber>30< / pat:ClaimNumber> <pat:ClaimText>30. The method of any one of claims 24-29, wherein the storage medium comprises 10% DMSO. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00031"> <pat:ClaimNumber>31< / pat:ClaimNumber> <pat:ClaimText>31. The method of any one of claims 24 to 30, wherein the storage medium comprises gentamicin at a concentration of at least 50 µg / mL. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00032"> <pat:ClaimNumber>32< / pat:ClaimNumber> <pat:ClaimText>32. The method of any one of claims 24 to 31, wherein the tumor tissue is first washed in Hank's Balanced Salt Solution (HBSS) before performing step (i). < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00033"> <pat:ClaimNumber>33< / pat:ClaimNumber> <pat:ClaimText>33. The method of claim 32, wherein the washing comprises three serial washes of at least three minutes each, with the HBSS replaced after each serial wash. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00034"> <pat:ClaimNumber>34< / pat:ClaimNumber> <pat:ClaimText>34. The method of any one of claims 24 to 33, wherein step (b) comprises immersing the vessel in water bath at a temperature of about 37°C for about 5 minutes. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00035"> <pat:ClaimNumber>35< / pat:ClaimNumber> <pat:ClaimText>35. The method of any one of claims 1-34, wherein the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, renal tumor tissue, pancreatic tumor tissue, glioblastoma tumor tissue, lung tumor tissue, colorectal tumor tissue, sarcoma tumor tissue, triple negative breast tumor tissue, cervical tumor tissue, ovarian tumor tissue, or HPV-positive tumor tissue. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00036"> <pat:ClaimNumber>36< / pat:ClaimNumber> <pat:ClaimText>36. The method of any one of claims 24-35, the method further comprising a split between step (c) and step (d). < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00037"> <pat:ClaimNumber>37< / pat:ClaimNumber> <pat:ClaimText>37. The method of claim 36, wherein the split occurs about 16 days after step (c) is initiated. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00038"> <pat:ClaimNumber>38< / pat:ClaimNumber> <pat:ClaimText>38. The method of any one of claims 24 to 37, wherein the first culture step is about 11 days. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00039"> <pat:ClaimNumber>39< / pat:ClaimNumber> <pat:ClaimText>39. The method of any one of claims 24 to 37, wherein steps (c) through (e) are performed over a period of about 22 days. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00040"> <pat:ClaimNumber>40< / pat:ClaimNumber> <pat:ClaimText>40. The method of any one of claims 24 to 37, wherein steps (c) through (e) are performed over a period of less than 20 days. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00041"> <pat:ClaimNumber>41< / pat:ClaimNumber> <pat:ClaimText>41. The method of any one of claims 24-40, wherein step (iv) comprises freezing the vessel at about -125 °C to about -150 °C. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00042"> <pat:ClaimNumber>42< / pat:ClaimNumber> <pat:ClaimText>42. The method of any one of claims 24-41, wherein step (v) comprises storing the vessel below about -130 °C. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00043"> <pat:ClaimNumber>43< / pat:ClaimNumber> <pat:ClaimText>43. The method of any one of claims 24-41, wherein step (v) comprises storing the vessel submerged in liquid nitrogen. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00044"> <pat:ClaimNumber>44< / pat:ClaimNumber> <pat:ClaimText>44. A use of expanded tumor infiltrating lymphocytes (TILs) for treating a subject with cancer, comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at 2 - 8°C for about 30 minutes; (iv) flash freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (b) thawing the tumor tissue; (c) adding the tumor fragments into a closed system; (d) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein 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 population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the therapeutic population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested TIL population from step (g) using a cryopreservation process; and (i) use of a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00045"> <pat:ClaimNumber>45< / pat:ClaimNumber> <pat:ClaimText>45. A use of expanded tumor infiltrating lymphocytes (TILs) in the manufacture of a medicament for treating a subject with cancer, comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at 2 - 8°C for about 30 minutes; (iv) flash freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (b) thawing the tumor tissue; (c) adding the tumor fragments into a closed system; (d) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein 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 population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the therapeutic population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested TIL population from step (g) using a cryopreservation process; and (i) use of a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00046"> <pat:ClaimNumber>46< / pat:ClaimNumber> <pat:ClaimText>46. The use of claim44 or 45, further comprising the steps of (i) concomitant use of a therapeutically effective dosage of aldesleukin or a biosimilar thereof to the subject and (k) concomitant use of a therapeutically effective dosage of a PD-1 / PD-L1 inhibitor to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00047"> <pat:ClaimNumber>47< / pat:ClaimNumber> <pat:ClaimText>47. The use of claim 46, wherein the PD-1 / PD-L1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, avelumab, durvalumab, atezolizumab, and biosimilars thereof. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00048"> <pat:ClaimNumber>48< / pat:ClaimNumber> <pat:ClaimText>48. The use of any one of claims 44-47, wherein the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck squamous cell cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, pancreatic cancer, and sarcoma. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00049"> <pat:ClaimNumber>49< / pat:ClaimNumber> <pat:ClaimText>49. A method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) providing tumor tissue from a patient; (b) incubating the tumor tissue for at least 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C; (c) flash freezing the tumor tissue; (d) storing the tumor tissue in a frozen state; (e) thawing the tumor tissue; (f) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (g) removing at least a plurality of the TILs; and (h) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00050"> <pat:ClaimNumber>50< / pat:ClaimNumber> <pat:ClaimText>50. A method for expanding TILs from frozen tumor tissue comprising: (a) providing tumor tissue from a patient; (b) trimming the tumor tissue to remove excess non-tumor tissue; (C) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) flash freezing the vessel; (f) storing the vessel such that the vessel remains frozen; (g) thawing the vessel; (h) treating the tumor tissue in a gas permeable container with a first cell culture medium and interleukin 2 (IL-2) to provide TILs; (i) removing at least a plurality of the TILs; and (j) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00051"> <pat:ClaimNumber>51< / pat:ClaimNumber> <pat:ClaimText>51. A use of expanded tumor infiltrating lymphocytes (TILs) for treating cancer for a human subject in need thereof, comprising use of the expanded tumor infiltrating lymphocytes (TILs) produced by a method comprising: (a) providing tumor tissue from the subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) flash freezing the vessel using the vapor phase of liquid nitrogen; (f) storing the vessel at the vapor phase of liquid nitrogen temperature; (g) thawing the tumor tissue; (h) adding the tumor tissue into a closed system; (i) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (h) to step (i) occurs without opening the system; (i) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (i) to step (j) occurs without opening the system; (k) harvesting the therapeutic population of TILs obtained from step (j), wherein the transition from step (j) to step (k) occurs without opening the system; (|) transferring the therapeutic population of TILs harvested in step (k) to an infusion bag, wherein the transfer from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (I) using a cryopreservation process; and (n) use of a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (m) to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00052"> <pat:ClaimNumber>52< / pat:ClaimNumber> <pat:ClaimText>52. A use of expanded tumor infiltrating lymphocytes (TILs) in the manufacture of a medicament for treating cancer for a human subject in need thereof, comprising use of the expanded tumor infiltrating lymphocytes (TILs) produced by a method comprising: (a) providing tumor tissue from the subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) flash freezing the vessel using the vapor phase of liquid nitrogen; (f) storing the vessel at the vapor phase of liquid nitrogen temperature; (g) thawing the tumor tissue; (h) adding the tumor tissue into a closed system; (i) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (h) to step (i) occurs without opening the system; (i) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (i) to step (j) occurs without opening the system; (k) harvesting the therapeutic population of TILs obtained from step (j), wherein the transition from step (j) to step (k) occurs without opening the system; (I) transferring the therapeutic population of TILs harvested in step (k) to an infusion bag, wherein the transfer from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (I) using a cryopreservation process; and (n) use of a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (m) to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00053"> <pat:ClaimNumber>53< / pat:ClaimNumber> <pat:ClaimText>53. A method for expanding tumor infiltrating lymphocytes (TILs) from frozen tumor tissue comprising: (a) providing tumor tissue from a patient, wherein the tumor tissue comprises TILs; (b) incubating the tumor tissue for about 30 minutes in a storage medium at a temperature in the range of about 2°C to about 8°C; (c) flash freezing the tumor tissue; (d) storing the tumor tissue in a frozen state: (e) thawing the tumor tissue; (f) treating the tumor tissue in a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 antibody to provide TILs; (g) removing at least a plurality of the TILs; and (h) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TlLs. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00054"> <pat:ClaimNumber>54< / pat:ClaimNumber> <pat:ClaimText>54. The method of claim 53, wherein the TILs in step (f) are cultured over a period of from about 3 to about 5 days, and wherein the TILs in step (h) are cultured over a period of from about 11 to about 13 days. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00055"> <pat:ClaimNumber>55< / pat:ClaimNumber> <pat:ClaimText>55. The method of claim 53, wherein the TILs in step (f) are cultured over a period of about 3 days, and wherein the TILs in step (h) are cultured over a period of about 13 days. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00056"> <pat:ClaimNumber>56< / pat:ClaimNumber> <pat:ClaimText>56. The method of claim 53, wherein the TILs in step (f) are cultured over a period of about 5 days, and wherein the TILs in step (h) are cultured over a period of about 11 days. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00057"> <pat:ClaimNumber>57< / pat:ClaimNumber> <pat:ClaimText>57. The method of any one of claims 53-56, further comprising (i) harvesting the TILs. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00058"> <pat:ClaimNumber>58< / pat:ClaimNumber> <pat:ClaimText>58. The method of claim 56 or 57, wherein the harvested TILs represent at least a 10000- fold increase in number. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00059"> <pat:ClaimNumber>59< / pat:ClaimNumber> <pat:ClaimText>59. A expanded tumor infiltrating lymphocytes (TILs) for use in treating a subject with cancer, comprising: (a) storing the tumor tissue in a frozen state, the method of storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable vessel containing a storage medium; (iii) incubating the vessel, which contains the tumor tissue and the storage medium, at 2 - 8°C for about 30 minutes; (iv) freezing the vessel using the vapor phase of liquid nitrogen; and (v) storing the vessel at the vapor phase of liquid nitrogen temperature; (b) thawing the tumor tissue; (c) adding the tumor fragments into a closed system; (d) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein 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 population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the system; (f) harvesting the therapeutic population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested TIL population from step (f) to an infusion bag, wherein the transfer from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag comprising the harvested TIL population from step (g) using a cryopreservation process; and (i) use of a therapeutically effective dosage of the third population of TILs from the infusion bag in step (h) to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00060"> <pat:ClaimNumber>60< / pat:ClaimNumber> <pat:ClaimText>60. The TILs for use of claim 59, further comprising the steps of (j) concomitant use of a therapeutically effective dosage of aldesleukin or a biosimilar thereof to the subject and (k) concomitant use of a therapeutically effective dosage of a PD-1 / PD-L1 inhibitor to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00061"> <pat:ClaimNumber>61< / pat:ClaimNumber> <pat:ClaimText>61. The TILs for use of claim 60, wherein the PD-1 / PD-L1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, avelumab, durvalumab, atezolizumab, and biosimilars thereof. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00062"> <pat:ClaimNumber>62< / pat:ClaimNumber> <pat:ClaimText>62. The TILs for use of any one of claims 59-61, wherein the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck squamous cell cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, pancreatic cancer, and sarcoma. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00063"> <pat:ClaimNumber>63< / pat:ClaimNumber> <pat:ClaimText>63. A expanded tumor infiltrating lymphocytes (TILs) for use in treating cancer for a human subject in need thereof, comprising use of the expanded tumor infiltrating lymphocytes (TILs) produced by a method comprising: (a) providing tumor tissue from the subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable vessel containing a storage medium; (d) incubating the vessel, which contains the tumor tissue and the storage medium, at a temperature in the range of about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) flash freezing the vessel using the vapor phase of liquid nitrogen; (f) storing the vessel at the vapor phase of liquid nitrogen temperature; (g) thawing the tumor tissue; (h) adding the tumor tissue into a closed system; (i) performing a first expansion by culturing a first population of TILs from the tumor tissue in a cell culture medium comprising 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, wherein the first expansion is performed for about 3-14 days to obtain the second population of TlLs, wherein the second population of TlLs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (h) to step (i) occurs without opening the system; (i) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (i) to step (j) occurs without opening the system; (k) harvesting the therapeutic population of TILs obtained from step (j), wherein the transition from step (j) to step (k) occurs without opening the system; (I) transferring the therapeutic population of TILs harvested in step (k) to an infusion bag, wherein the transfer from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag comprising the therapeutic population of TILs from step (I) using a cryopreservation process; and (n) use of a therapeutically effective dosage of the therapeutic population of TILs from the infusion bag in step (m) to the subject. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00064"> <pat:ClaimNumber>64< / pat:ClaimNumber> <pat:ClaimText>64. The method of any one of claims 1-22, wherein the tumor fragments are incubated in the cryopreservation medium for 30 minutes to 70 minutes. < / pat:ClaimText> < / pat:Claim> <pat:Claim com:id="CLM-00065"> <pat:ClaimNumber>65< / pat:ClaimNumber> <pat:ClaimText>65. The method of any one of claims 1-22, wherein the tumor fragments are incubated in the cryopreservation medium for 30 minutes to 60 minutes. < / pat:ClaimText> < / pat:Claim> < / pat:Claims>