Expansion of TILs from cryopreserved tumor samples

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international PCT application claims priority to U.S. Provisional Patent Application No. 62 / 733,937, filed September 20, 2018, and U.S. Provisional Patent Application No. 62 / 879,881, filed July 29, 2019, each of which is incorporated by reference in its entirety into this specification.

[0002] FIELD OF THE INVENTION

[0002] The invention described herein relates generally to the expansion of lymphocytes, and more particularly, but not exclusively, to the expansion of lymphocytes from cryopreserved tumor tissue. [Background technology]

[0003] Background of the Invention

[0003] Treating resistant cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) may be a powerful approach to treating patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires large quantities of TILs, thus necessitating a robust and reliable process for manufacturing and commercialization. This scaling for commercialization presents serious challenges due to numerous technical, logistical, and regulatory issues associated with cell expansion. IL-2-based TIL expansion followed by the "rapid expansion process" (REP) is becoming the preferred method for TIL expansion due to 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. REP can result in a 1,000-fold expansion of TILs in a 14-day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) as feeder cells, often from multiple donors, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2. (Dudley, et al., J. Immunother. 2003, 26, 332-42) TILs that have undergone the REP procedure have produced successful adoptive cell therapy in some melanoma patients after host immunosuppression.

[0004] Current TIL manufacturing processes are limited by time, expense, sterility concerns, and other factors described herein, significantly limiting the potential for commercializing such processes. Among the many limitations of current processes is a reliance on fresh tumor tissue to initiate manufacturing, which often necessitates expensive, costly local manufacturing facilities due to the difficulty of transporting fresh tumors long distances. There is an urgent need to provide TIL manufacturing processes and therapies that do not rely on fresh tumor tissue, are amenable to commercial-scale manufacturing, and are suitable for regulatory approval for use in geographically widely dispersed human patients. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention

[0005] In one embodiment, the present invention provides a method of TIL expansion utilizing frozen tumor tissue as the starting TIL source for producing a therapeutic TIL population.

[0006]

[0006] In one embodiment, the present invention provides (i) Fragmenting tumor tissue; (ii) incubating the fragments in cryopreservation medium; and (iii) Freezing the fragments, the freezing being flash freezing using the vapor phase of liquid nitrogen. The present invention provides a method for cryopreserving tumor tissue for the production of tumor infiltrating lymphocytes (TILs), comprising:

[0007]

[0007] In one embodiment, the present invention provides (i) fragmenting the tumor sample; (ii) incubating the fragments in cryopreservation medium; and (iii) freezing the fragments, the freezing being flash freezing using the vapor phase of liquid nitrogen; The present invention provides cryopreserved tumor fragments for the production of tumor-infiltrating lymphocytes (TILs), prepared by a process comprising:

[0008]

[0008] In one embodiment, the present invention provides (i) fragmenting the tumor sample; (ii) incubating the fragments in a cryopreservation medium containing about 10% v / v dimethyl sulfoxide at a temperature ranging from about 2°C to about 8°C for about 20 minutes to about 70 minutes; and (iii) freezing the fragments, the freezing being flash freezing using the vapor phase of liquid nitrogen; The present invention provides cryopreserved tumor fragments for the production of tumor-infiltrating lymphocytes (TILs), prepared by a process comprising:

[0009]

[0009] In one embodiment, the present invention provides (a) storing tumor tissue in a frozen state, wherein the method of storing the tumor tissue comprises incubating the tumor tissue in a storage medium at a temperature ranging from about 2°C to about 8°C for at least 30 minutes prior to freezing; (b) thawing the tumor tissue; (c) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (d) removing at least a plurality of TILs; and (e) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0010]

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

[0011] In some embodiments, the storage medium comprises 2-12% v / v (volume:volume) dimethyl sulfoxide (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 5-10% 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 2-12% w / w (weight:weight) dimethyl sulfoxide (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 5-10% 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 in storage medium at a temperature ranging from about 2°C to about 8°C for at least 30 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for 30 minutes to about 60 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 45 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 60 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for 60 minutes before freezing.

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

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

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

[0017] In some embodiments, the fresh tumor tissue is trimmed to a diameter of 1.5 mm to 6 mm and a thickness of 1.5 mm to 6 mm. In yet other embodiments, the fresh tumor tissue is trimmed to approximately 6 mm x 6 mm x 6 mm.

[0018]

[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 at about 0.25 μg / mL to about 2.5 μg / mL. In some embodiments, the antifungal agent is fungin and is present at about 5 μg / mL to about 50 μg / mL.

[0019] In some embodiments, the fresh tumor tissue is first washed with Hank's Balanced Salt Solution (HBSS) or another suitable medium or solution prior to performing the freezing step of the method. In some embodiments, the washing comprises at least three successive washes of at least 3 minutes each, with the HBSS being replaced after each wash.

[0020]

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

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

[0022]

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

[0023]

[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 splitting between the first expansion culture step (c) and the second expansion culture step (e). In one embodiment, the splitting occurs on day 16. In some embodiments, the first culture step is completed in about 11 days. In other embodiments, the first expansion culture (or first culture) step is completed in about 7 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 7 days and the second expansion culture (or second culture) step is completed in about 14 days. In some embodiments, steps (c) through (e) are completed in about 22 days. In yet other embodiments, steps (c) through (e) are completed in 21 days; in some embodiments, steps (c) through (e) are completed in about 20 days; and in some embodiments, steps (c) through (e) are completed in about 16 days.

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

[0026] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) obtaining tumor tissue from a subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable container containing a storage medium; (d) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) freezing the container using the vapor phase of liquid nitrogen; (f) storing the container in the vapor phase at liquid nitrogen temperatures; (g) thawing the tumor tissue; (h) adding tumor tissue to a closed system; (i) performing a first expansion culture by culturing a first population of TILs from the tumor tissue in a cell culture medium having IL-2 to generate a second population of TILs, wherein the first expansion culture is performed in a closed container that provides a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second population of TILs, the second population of TILs being at least 50-fold more numerous than the first population of TILs, and the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (i) to step (j) occurs without opening the system; (k) recovering the therapeutic TIL population obtained from step (j), wherein the transition from step (j) to step (k) occurs without releasing the system; (l) transferring the therapeutic TIL population collected in step (k) to an infusion bag, wherein the transition from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag containing the therapeutic TIL population from step (l) using a cryopreservation process; and (n) administering a therapeutically effective dose of the therapeutic TIL population to the subject from the infusion bag in step (m). The method includes administering expanded tumor-infiltrating lymphocytes (TILs), comprising:

[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, resistant melanoma, metastatic melanoma, and non-small cell lung cancer. The histology of solid tumors comprises interdependent tissue compartments, including parenchyma (cancer cells), and supporting stromal cells in which cancer cells may disperse and provide a supportive microenvironment.

[0028]

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

[0029]

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

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

[0031] In some embodiments, the fresh tumor tissue is first washed with Hank's Balanced Salt Solution (HBSS) before performing the freezing step of the method. In some embodiments, the washing comprises at least three successive washes of at least 3 minutes each, and the HBSS is replaced after each wash.

[0032]

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

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

[0034]

[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 splitting between the first expansion culture (or first culture) step and the second expansion culture (or second culture) step. In one embodiment, the splitting occurs on day 16. In some embodiments, the first culture step is completed in about 11 days. In other embodiments, the first expansion culture (or first culture) step is completed in about 7 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 7 days, and the second expansion culture (or second culture) step is completed in about 14 days. In some embodiments, steps (d) to (f) or steps (i) to (k), as applicable, are completed in about 22 days. In still other embodiments, steps (d) through (f) or steps (i) through (k), as applicable, are completed in about 21 days; in some embodiments, steps (d) through (f) or steps (i) through (k), as applicable, are completed in about 20 days; in some embodiments, steps (d) through (f) or steps (i) through (k), as applicable, are completed in about 16 days.

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

[0037] In some embodiments, the fresh tumor tissue is first washed with Hank's Balanced Salt Solution (HBSS) before performing the freezing step of the method. In some embodiments, the washing comprises at least three successive washes of at least 3 minutes each, and the HBSS is replaced after each wash.

[0038]

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

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

[0040]

[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 splitting between the first and second expansion steps. In one embodiment, the splitting occurs on day 16. In some embodiments, the first expansion step is completed in about 11 days. In other embodiments, the first expansion step is completed in about 7 days. In still other embodiments, the first expansion step is completed in about 7 days and the second expansion step is completed in about 14 days. In some embodiments, steps (e) through (g) are completed in about 22 days. In still other embodiments, steps (e) through (g) are completed in about 21 days; in some embodiments, steps (e) through (g) are completed in about 20 days; and in some embodiments, steps (e) through (g) are completed in about 16 days.

[0042]

[0042] In some embodiments, the expansion culture method according to the present invention is completed in about 16 days. In some embodiments, the expansion culture method includes a first expansion culture step (pre-REP step) and a second expansion culture step (REP step). In one embodiment, the first expansion culture step is about 3 to about 5 days. In another embodiment, the first expansion culture step is about 3 days. In another embodiment, the first expansion culture step is about 5 days. In some embodiments, the second expansion culture step is about 11 to about 13 days. In another embodiment, the second expansion culture step is about 11 days. In another embodiment, the second expansion culture step is about 13 days. In some embodiments, the first expansion culture step is about 5 days and the second expansion culture step is about 11 days. In some embodiments, the first expansion culture step is about 3 days and the second expansion culture 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 day 11, and the TILs are split on day 5 of the second expansion step. In some embodiments, the second expansion step is about day 13, and the TILs are split on day 6 of the second expansion step.

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

[0045] In some embodiments, feeder cells are used during the second expansion step. In one embodiment, in a 1 / 100 scale process, about 2-50×10 6 In another embodiment, about 25 x 10 feeder cells are used during the second expansion step. 6 In another embodiment, about 50 x 10 feeder cells are used. 6 In some embodiments, a full-scale process uses about 2-50 x 10 feeder cells. 9 In some embodiments, a full-scale process uses about 2.5 x 10 feeder cells. 9 or approximately 5 x 10 9 In some embodiments, feeder cells are used. In some embodiments, the feeder cells are PBMCs.

[0046] In some embodiments, the second expansion step comprises culturing at least about 5×10 6 ~About 200×10 6In 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]

[0047] Other embodiments include combinations and variations of these compositions and methods.

[0048] 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. [Brief explanation of the drawings]

[0049] [Figure 1]

[0049] Figure 2 shows the main steps of an embodiment of process 2A, including, optionally, cryopreservation of fresh tumor tissue after fragmentation in step B. The total time from the start of the first expansion culture to the end of step E is approximately 22 days. [Figure 2A]

[0050] Panels A-C show various steps of an embodiment of the TIL manufacturing process, including cryopreservation of fresh tumor fragments to allow for later initiation of pre-REP cultures. [Figure 2B] Panels A-C show various steps of an embodiment of the TIL manufacturing process, including cryopreservation of fresh tumor fragments to allow for later initiation of pre-REP cultures. [Figure 2C] Panels A-C show various steps of an embodiment of the TIL manufacturing process, including cryopreservation of fresh tumor fragments to allow for later initiation of pre-REP cultures. [Figure 3]

[0051] 1 illustrates an embodiment of TIL production involving cryopreservation of fresh tumor fragments that are subsequently used to initiate pre-REP cultures. [Figure 4]

[0052] 1 illustrates an embodiment of TIL production involving cryopreservation of fresh tumor fragments that are subsequently used to initiate pre-REP cultures. [Figure 5]

[0053] In general, compare the embodiment of Process 1C with an exemplary embodiment of Process 2A, which contemplates initiation of pre-REP cultures from either fresh tumor tissue fragments or thawed cryopreserved tumor tissue fragments. [Figure 6]

[0054] Additionally, the embodiment of Process 1C is compared to the embodiment of Process 2A. [Figure 7]

[0055] 1 illustrates an embodiment of a 2A process in which REP is started "early." [Figure 8]

[0056] The total number of viable cells from the Gen2A process and a modified Gen2A process called "Gen2.2" were compared, where the pre-REP culture was initiated fresh, flash-frozen, or frozen after 30 or 60 minutes of incubation in storage medium at 2°C to 8°C. Cell counts were taken at the end of the pre-REP culture. [Figure 9]

[0057] We compared the total viable cell counts from the Gen2A process and a modified Gen2A process called "Gen2.2," where the pre-REP culture was initiated fresh, flash-frozen, or frozen after 30 or 60 minutes of incubation in storage medium at 2°C to 8°C. Cell counts were at the end of the REP culture. [Figure 10]

[0058] We compare IFN-γ produced by cells derived from the Gen2A process and a modified Gen2A process called "Gen2.2," where pre-REP cultures were initiated fresh, flash-frozen, or frozen after 30 or 60 minutes of incubation in storage medium at 2°C to 8°C. [Figure 11]

[0059] 1 shows the main steps of a Gen2 embodiment compared to the main steps of a Gen2.2 embodiment. [Figure 12]

[0060] The main steps of the TIL production process are shown at 1 / 100 scale. [Figure 13A]

[0061] Figures 13A–13E show the total viable cells (TVC) of tumor samples processed and collected at pre-REP from five different patients. TVC was measured using fresh (Gen2 control) and frozen tumor samples. TVC was recorded on day 11 for fresh tumor samples and on day 7 for frozen tumor samples. Figure 13A (patient M1125) shows the only patient whose tumor sample was subjected to different freezing methods: flash freezing in the vapor phase of liquid nitrogen and incubation at 2–8°C for 30 or 60 minutes before flash freezing in the vapor phase of liquid nitrogen. For the remaining patients, S13018, OV8022, T6042, and O8026 (Figures 13B–13E, respectively), tumor samples were incubated at 2–8°C for 60 minutes before flash freezing. For each patient, tumor samples were cut into 3 mm diameter fragments, and 4–6 fragments were used for culture. Each bar on the graph represents the TVC extrapolated to full scale (multiplied by 10). In Figure 13A, frozen early pre-REP cultures yielded a TVC of approximately 1% of that of fresh, in Figure 13B, approximately 25% of that of fresh, in Figure 13C, approximately 200% of that of fresh, in Figure 13D, approximately 96% of that of fresh, and in Figure 13E, approximately 3% of that of fresh. The data show that the TVC from day 7 frozen tumor samples is equivalent to that from day 11 fresh tumor samples. [Figure 13B]Figures 13A-13E show the total viable cells (TVC) of tumor samples processed and collected at pre-REP from five different patients. TVC was measured using fresh (Gen2 control) and frozen tumor samples. TVC was recorded on day 11 for fresh tumor samples and on day 7 for frozen tumor samples. Figure 13A (patient M1125) shows the only patient whose tumor sample was subjected to different freezing methods: flash freezing in the vapor phase of liquid nitrogen and incubation at 2-8°C for 30 or 60 minutes before flash freezing in the vapor phase of liquid nitrogen. For the remaining patients, S13018, OV8022, T6042, and O8026 (Figures 13B-13E, respectively), tumor samples were incubated at 2-8°C for 60 minutes before flash freezing. For each patient, tumor samples were cut into 3 mm diameter fragments, and 4-6 fragments were used for culture. Each bar on the graph represents the TVC extrapolated to full scale (multiplied by 10). In Figure 13A, frozen early pre-REP cultures yielded a TVC of approximately 1% of that of fresh, in Figure 13B, approximately 25% of that of fresh, in Figure 13C, approximately 200% of that of fresh, in Figure 13D, approximately 96% of that of fresh, and in Figure 13E, approximately 3% of that of fresh. The data show that the TVC from day 7 frozen tumor samples is equivalent to that from day 11 fresh tumor samples. [Figure 13C]Figures 13A-13E show the total viable cells (TVC) of tumor samples processed and collected at pre-REP from five different patients. TVC was measured using fresh (Gen2 control) and frozen tumor samples. TVC was recorded on day 11 for fresh tumor samples and on day 7 for frozen tumor samples. Figure 13A (patient M1125) shows the only patient whose tumor sample was subjected to different freezing methods: flash freezing in the vapor phase of liquid nitrogen and incubation at 2-8°C for 30 or 60 minutes before flash freezing in the vapor phase of liquid nitrogen. For the remaining patients, S13018, OV8022, T6042, and O8026 (Figures 13B-13E, respectively), tumor samples were incubated at 2-8°C for 60 minutes before flash freezing. For each patient, tumor samples were cut into 3 mm diameter fragments, and 4-6 fragments were used for culture. Each bar on the graph represents the TVC extrapolated to full scale (multiplied by 10). In Figure 13A, frozen early pre-REP cultures yielded a TVC of approximately 1% of that of fresh, in Figure 13B, approximately 25% of that of fresh, in Figure 13C, approximately 200% of that of fresh, in Figure 13D, approximately 96% of that of fresh, and in Figure 13E, approximately 3% of that of fresh. The data show that the TVC from day 7 frozen tumor samples is equivalent to that from day 11 fresh tumor samples. [Figure 13D]Figures 13A-13E show the total viable cells (TVC) of tumor samples processed and collected at pre-REP from five different patients. TVC was measured using fresh (Gen2 control) and frozen tumor samples. TVC was recorded on day 11 for fresh tumor samples and on day 7 for frozen tumor samples. Figure 13A (patient M1125) shows the only patient whose tumor sample was subjected to different freezing methods: flash freezing in the vapor phase of liquid nitrogen and incubation at 2-8°C for 30 or 60 minutes before flash freezing in the vapor phase of liquid nitrogen. For the remaining patients, S13018, OV8022, T6042, and O8026 (Figures 13B-13E, respectively), tumor samples were incubated at 2-8°C for 60 minutes before flash freezing. For each patient, tumor samples were cut into 3 mm diameter fragments, and 4-6 fragments were used for culture. Each bar on the graph represents the TVC extrapolated to full scale (multiplied by 10). In Figure 13A, frozen early pre-REP cultures yielded a TVC of approximately 1% of that of fresh, in Figure 13B, approximately 25% of that of fresh, in Figure 13C, approximately 200% of that of fresh, in Figure 13D, approximately 96% of that of fresh, and in Figure 13E, approximately 3% of that of fresh. The data show that the TVC from day 7 frozen tumor samples is equivalent to that from day 11 fresh tumor samples. [Figure 13E]Figures 13A-13E show the total viable cells (TVC) of tumor samples processed and collected at pre-REP from five different patients. TVC was measured using fresh (Gen2 control) and frozen tumor samples. TVC was recorded on day 11 for fresh tumor samples and on day 7 for frozen tumor samples. Figure 13A (patient M1125) shows the only patient whose tumor sample was subjected to different freezing methods: flash freezing in the vapor phase of liquid nitrogen and incubation at 2-8°C for 30 or 60 minutes before flash freezing in the vapor phase of liquid nitrogen. For the remaining patients, S13018, OV8022, T6042, and O8026 (Figures 13B-13E, respectively), tumor samples were incubated at 2-8°C for 60 minutes before flash freezing. For each patient, tumor samples were cut into 3 mm diameter fragments, and 4-6 fragments were used for culture. Each bar on the graph represents the TVC extrapolated to full scale (multiplied by 10). In Figure 13A, frozen early pre-REP cultures yielded a TVC of approximately 1% of that of fresh, in Figure 13B, approximately 25% of that of fresh, in Figure 13C, approximately 200% of that of fresh, in Figure 13D, approximately 96% of that of fresh, and in Figure 13E, approximately 3% of that of fresh. The data show that the TVC from day 7 frozen tumor samples is equivalent to that from day 11 fresh tumor samples. [Figure 14A]

[0062] Figures 14A-14E show TVCs of tumor samples from the same five patients as Figure 13 collected during Early REP. As shown in Figure 13, cells were harvested on day 11 (fresh) and day 7 (frozen). In Figure 14A, the frozen Early REP cultures yielded approximately 86% TVC relative to fresh; in Figure 14B, approximately 98% TVC relative to fresh; in Figure 14C, approximately 124% TVC relative to fresh; in Figure 14D, approximately 286% TVC relative to fresh; and in Figure 14E, approximately 167% TVC relative to fresh. The data show that the overall yield (measured as TVC) from frozen tumor samples on day 7 was comparable to that from fresh tumor samples on day 11 during Early REP. [Figure 14B]Figures 14A-14E show TVCs of tumor samples from the same five patients as Figure 13 collected during Early REP. As shown in Figure 13, cells were harvested on day 11 (fresh) and day 7 (frozen). In Figure 14A, the frozen Early REP cultures yielded approximately 86% TVC relative to fresh; in Figure 14B, approximately 98% TVC relative to fresh; in Figure 14C, approximately 124% TVC relative to fresh; in Figure 14D, approximately 286% TVC relative to fresh; and in Figure 14E, approximately 167% TVC relative to fresh. The data show that the overall yield (measured as TVC) from frozen tumor samples on day 7 was comparable to that from fresh tumor samples on day 11 during Early REP. [Figure 14C] Figures 14A-14E show TVCs of tumor samples from the same five patients as Figure 13 collected during Early REP. As shown in Figure 13, cells were harvested on day 11 (fresh) and day 7 (frozen). In Figure 14A, the frozen Early REP cultures yielded approximately 86% TVC relative to fresh; in Figure 14B, approximately 98% TVC relative to fresh; in Figure 14C, approximately 124% TVC relative to fresh; in Figure 14D, approximately 286% TVC relative to fresh; and in Figure 14E, approximately 167% TVC relative to fresh. The data show that the overall yield (measured as TVC) from frozen tumor samples on day 7 was comparable to that from fresh tumor samples on day 11 during Early REP. [Figure 14D] Figures 14A-14E show TVCs of tumor samples from the same five patients as Figure 13 collected during Early REP. As shown in Figure 13, cells were harvested on day 11 (fresh) and day 7 (frozen). In Figure 14A, the frozen Early REP cultures yielded approximately 86% TVC relative to fresh; in Figure 14B, approximately 98% TVC relative to fresh; in Figure 14C, approximately 124% TVC relative to fresh; in Figure 14D, approximately 286% TVC relative to fresh; and in Figure 14E, approximately 167% TVC relative to fresh. The data show that the overall yield (measured as TVC) from frozen tumor samples on day 7 was comparable to that from fresh tumor samples on day 11 during Early REP. [Figure 14E]Figures 14A-14E show TVCs of tumor samples from the same five patients as Figure 13 collected during Early REP. As shown in Figure 13, cells were harvested on day 11 (fresh) and day 7 (frozen). In Figure 14A, the frozen Early REP cultures yielded approximately 86% TVC relative to fresh; in Figure 14B, approximately 98% TVC relative to fresh; in Figure 14C, approximately 124% TVC relative to fresh; in Figure 14D, approximately 286% TVC relative to fresh; and in Figure 14E, approximately 167% TVC relative to fresh. The data show that the overall yield (measured as TVC) from frozen tumor samples on day 7 was comparable to that from fresh tumor samples on day 11 during Early REP. [Figure 15]

[0063] Figure 15 shows the CD8 / CD4 ratios of fresh and frozen tumor samples from four patients. Fresh and 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-Cy7, and CD16-APC-Vio770). TIL identity was determined based on TCRa / 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 showed high CD8 expression, but overall the CD8 / CD4 ratios were comparable between fresh and frozen tumor samples. [Figure 16A]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 16B]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 16C]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 16D]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 16E]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 16F]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 16G]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 16H]

[0064] Figures 16A-16H show the expression of CD3+CD45+ and other markers in the same four patients as in Figure 15. Figures 16A and 16B show data for patient M1125. Figures 16C and 16D show data for patient S13018. Figures 16E and 16F show data for patient OV8022. Figures 16G and 16H show data for patient T6042. The data show that the purity of TILs was comparable between fresh and frozen tumor samples. [Figure 17A]

[0065] Figures 17A-17C show the expression of CD28, CD27, and CD57 in cells expressing CD4 or CD8 from fresh and frozen tumor samples. Figure 17A relates to patient M1125. [Figure 17B] Figure 17B relates to patient S13018. [Figure 17C] Figure 17C relates to patient T6042. The data show that the differentiation state between fresh and frozen tumor samples is comparable. [Figure 18A]

[0066] Figures 18A-18C show the memory status of fresh and frozen tumor samples for cells expressing CD4 or CD8. Figure 18A relates to patient M1125. [Figure 18B] Figure 18B relates to patient S13018. [Figure 18C] Figure 18C relates to patient T6042. The data demonstrate comparable memory status between fresh and frozen tumor samples. TN: naive (CD45RA+CCR7+), TCM: central memory (CD45RA-CCR7+), TEM: effector memory (CD45RA-CCR7-), TEMRA / TEFF: RA+ effector memory / effector (CD45RA+CCR7), central memory / activated cells (CD45RA+CD62L+). Bar graphs shown are the percentage of positive CD45+ / -, CCR7+ / -, or CD62L+ / - cells when gated on CD4+ or CD8+. [Figure 19A]

[0067] Figures 19A-19C show activation / depletion markers in fresh and frozen tumor samples for cells expressing CD4 (upper panels) or CD8 (lower panels) in three patients: M1125 (Figure 19A); S13018 (Figure 19B); and T6042 (Figure 19C). Activation and depletion of REP TILs were determined by multicolor flow cytometry. Tumor tissues were 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, and LAG-3-APC-eFluor 780) and expression was measured using flow cytometry. Overall, TIL activation / depletion status is comparable between fresh and frozen tumor samples. [Figure 19B] Figures 19A-19C show activation / depletion markers in fresh and frozen tumor samples for cells expressing CD4 (upper panels) or CD8 (lower panels) from three patients: M1125 (Figure 19A); S13018 (Figure 19B); and T6042 (Figure 19C). Activation and depletion of REP TILs were determined by multicolor flow cytometry. Tumor tissues were 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, and LAG-3-APC-eFluor 780) and expression was measured using flow cytometry. Overall, TIL activation / depletion status is comparable between fresh and frozen tumor samples. [Figure 19C] Figures 19A-19C show activation / depletion markers in fresh and frozen tumor samples for cells expressing CD4 (upper panels) or CD8 (lower panels) from three patients: M1125 (Figure 19A); S13018 (Figure 19B); and T6042 (Figure 19C). Activation and depletion of REP TILs were determined by multicolor flow cytometry. Tumor tissues were 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, and LAG-3-APC-eFluor 780) and expression was measured using flow cytometry. Overall, TIL activation / depletion status is comparable between fresh and frozen tumor samples. [Figure 20A]

[0068] Figures 20A-20C show IFNγ function in fresh and frozen tumor samples from three patients: M1125 (Figure 20A); S13018 (Figure 20B); and T6042 (Figure 20C). 1 x 105 REP TILs were plated on anti-CD3 plates for 24 hours. Culture supernatants were collected, and IFNγ cytokines were measured using an IFNγ Qantikine ELISA kit. Bar graphs shown here are the mean + SD of triplicate measurements. Statistical significance was calculated using Student's "t" test. *P<0.05, **P<0.01, ***P<0.001. Overall, the data indicate that IFNγ function was higher in frozen tumor samples. [Figure 20B]Figures 20A-20C show IFNγ function in fresh and frozen tumor samples from three patients: M1125 (Figure 20A); S13018 (Figure 20B); and T6042 (Figure 20C). 1x105 REP TILs were plated on anti-CD3 plates for 24 hours. Culture supernatants were collected, and IFNγ cytokines were measured using an IFNγ Qantikine ELISA kit. Bar graphs shown here are the mean + SD of triplicate measurements. Statistical significance was calculated using Student's "t" test. *P<0.05, **P<0.01, ***P<0.001. Overall, the data indicate that IFNγ function was higher in frozen tumor samples. [Figure 20C] Figures 20A-20C show IFNγ function in fresh and frozen tumor samples from three patients: M1125 (Figure 20A); S13018 (Figure 20B); and T6042 (Figure 20C). 1x105 REP TILs were plated on anti-CD3 plates for 24 hours. Culture supernatants were collected, and IFNγ cytokines were measured using an IFNγ Qantikine ELISA kit. Bar graphs shown here are the mean + SD of triplicate measurements. Statistical significance was calculated using Student's "t" test. *P<0.05, **P<0.01, ***P<0.001. Overall, the data indicate that IFNγ function was higher in frozen tumor samples. [Figure 21A]

[0069] Figures 21A-21C show granzyme B (GzmB) function in fresh and frozen tumor samples from three patients: M1125 (Figure 21A); S13018 (Figure 21B); and T6042 (Figure 21C). 1x105 REP TILs were plated on anti-CD3 plates for 24 hours. Culture supernatants were collected and measured for granzyme B using a Duo Set ELISA kit. Bar graphs shown here are the mean + SD of triplicate determinations. Statistical significance was calculated using Student's "t" test. *P<0.05, **P<0.01, ***P<0.001. Overall, the data indicate that granzyme B (GzmB) function is comparable / higher in frozen tumor samples. [Figure 21B] Figures 21A-21C show granzyme B (GzmB) function in fresh and frozen tumor samples from three patients: M1125 (Figure 21A); S13018 (Figure 21B); and T6042 (Figure 21C). 1x105 REP TILs were plated on anti-CD3 plates for 24 hours. Culture supernatants were collected and measured for granzyme B using a Duo Set ELISA kit. Bar graphs shown here are the mean + SD of triplicate determinations. Statistical significance was calculated using Student's "t" test. *P<0.05, **P<0.01, ***P<0.001. Overall, the data indicate that granzyme B (GzmB) function is comparable / higher in frozen tumor samples. [Figure 21C] Figures 21A-21C show granzyme B (GzmB) function in fresh and frozen tumor samples from three patients: M1125 (Figure 21A); S13018 (Figure 21B); and T6042 (Figure 21C). 1x105 REP TILs were plated on anti-CD3 plates for 24 hours. Culture supernatants were collected and measured for granzyme B using a Duo Set ELISA kit. Bar graphs shown here are the mean + SD of triplicate determinations. Statistical significance was calculated using Student's "t" test. *P<0.05, **P<0.01, ***P<0.001. Overall, the data indicate that granzyme B (GzmB) function is comparable / higher in frozen tumor samples. [Figure 22A]

[0070] Figures 22A-22C show CD107A expression in fresh and frozen tumor samples from three patients: M1125 (Figure 22A); S13018 (Figure 22B); and T6042 (Figure 22C). 2 x 10 REP TILs were stimulated with or without PMA / IO for 2 hours, stained with CD107A-APC700, and analyzed by flow cytometry. Bar graphs represent CD107A-positive cells gated on CD8 or CD4. Overall, the data show that CD107A expression is comparable between fresh and frozen tumor samples. [Figure 22B]Figures 22A-22C show CD107A expression in fresh and frozen tumor samples from three patients: M1125 (Figure 22A); S13018 (Figure 22B); and T6042 (Figure 22C). 2x105 REP TILs were stimulated with or without PMA / IO for 2 hours, stained with CD107A-APC700, and analyzed by flow cytometry. Bar graphs represent CD107A-positive cells gated on CD8 or CD4. Overall, the data show that CD107A expression is comparable between fresh and frozen tumor samples. [Figure 22C] Figures 22A-22C show CD107A expression in fresh and frozen tumor samples from three patients: M1125 (Figure 22A); S13018 (Figure 22B); and T6042 (Figure 22C). 2x105 REP TILs were stimulated with or without PMA / IO for 2 hours, stained with CD107A-APC700, and analyzed by flow cytometry. Bar graphs represent CD107A-positive cells gated on CD8 or CD4. Overall, the data show that CD107A expression is comparable between fresh and frozen tumor samples. [Figure 23A]

[0071] Figures 23A-23C show telomere length relative to 1301 cells in fresh and frozen tumor samples from three patients: M1125 (Figure 23A); S13018 (Figure 23B); and T6042 (Figure 23C). Flow FISH was performed using a Dako / Agilent Pathology Solutions (Telomere PNA Kit / FITC for Flow Cytometry) kit, and the average telomere repeat length was measured according to the manufacturer's instructions. The 1301 T-cell leukemia cell line (Sigma-Aldrich, St. Louis, MO) was used as an internal reference standard for each assay. Individual TILs were counted and mixed with 1301 cells at a 1:1 cell ratio. 2 × 10 TILs were mixed with 2 × 10 1301 cells, and in situ hybridization was performed using a FITC-conjugated telomere PNA probe (FITC-00-CCCTAA-CCC-TAA-CCC-TAA) and analyzed using a flow cytometer (BD Canto II). Telomere fluorescence of test samples was expressed as a percentage of the geometric mean fluorescence (fl) of 1301 cells according to 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 show that telomere length is comparable to or greater in frozen tumor samples than in fresh tumor samples. [Figure 23B]Figures 23A-23C show telomere length relative to 1301 cells in fresh and frozen tumor samples from three patients: M1125 (Figure 23A); S13018 (Figure 23B); and T6042 (Figure 23C). Flow FISH was performed using a Dako / Agilent Pathology Solutions (Telomere PNA Kit / FITC for Flow Cytometry) kit, and the average length of telomere repeats was measured according to the manufacturer's instructions. The 1301 T-cell leukemia cell line (Sigma-Aldrich, St. Louis, MO) was used as an internal reference standard for each assay. Individual TILs were counted and mixed with 1301 cells at a 1:1 cell ratio. 2 × 10 TILs were mixed with 2 × 10 1301 cells, and in situ hybridization was performed using a FITC-conjugated telomere PNA probe (FITC-00-CCCTAA-CCC-TAA-CCC-TAA) and analyzed using a flow cytometer (BD Canto II). Telomere fluorescence of test samples was expressed as a percentage of the geometric mean fluorescence (fl) of 1301 cells according to 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 show that telomere length is comparable to or greater in frozen tumor samples than in fresh tumor samples. [Figure 23C]Figures 23A-23C show telomere length relative to 1301 cells in fresh and frozen tumor samples from three patients: M1125 (Figure 23A); S13018 (Figure 23B); and T6042 (Figure 23C). Flow FISH was performed using a Dako / Agilent Pathology Solutions (Telomere PNA Kit / FITC for Flow Cytometry) kit, and the average length of telomere repeats was measured according to the manufacturer's instructions. The 1301 T-cell leukemia cell line (Sigma-Aldrich, St. Louis, MO) was used as an internal reference standard for each assay. Individual TILs were counted and mixed with 1301 cells at a 1:1 cell ratio. 2 × 10 TILs were mixed with 2 × 10 1301 cells, and in situ hybridization was performed using a FITC-conjugated telomere PNA probe (FITC-00-CCCTAA-CCC-TAA-CCC-TAA) and analyzed using a flow cytometer (BD Canto II). Telomere fluorescence of test samples was expressed as a percentage of the geometric mean fluorescence (fl) of 1301 cells according to 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 show that telomere length is comparable to or greater in frozen tumor samples than in fresh tumor samples. [Figure 24]

[0072] FIG. 24 is a flow chart showing specific embodiments of the expansion process of TILs from fresh and frozen tumor fragments: Fresh Method (“GEN2”), Early REP Method 1 (“ER-1”), and Early REP Method 2 (“ER-2”). [Figure 25]

[0073] Figure 25 shows structures IA and IB, which contain three linearly linked TNFRSF binding domains, e.g., from an antibody that binds to 4-1BBL or 4-1BB, that fold to form a trivalent protein, then linked to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link the two trivalent proteins via a disulfide bond (small elongated oval), stabilizing the structure and providing an agonist that can combine six receptor intracellular signaling domains and signaling proteins to form a signaling complex. DETAILED DESCRIPTION OF THE INVENTION

[0050] Brief Description of Sequence Listing

[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 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 recombinant human IL-4 protein.

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

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

[0081] SEQ ID NO: 8 is the amino acid sequence of 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 mouse 4-1BB.

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

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

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

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

[0088] SEQ ID NO: 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

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

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

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

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

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

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

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

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

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

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

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

[0100] SEQ ID NO: 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0101] SEQ ID NO: 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0102] SEQ ID NO: 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0103] SEQ ID NO: 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0104] SEQ ID NO: 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0105] SEQ ID NO: 32 is the linker of the TNFRSF agonist fusion protein.

[0106] SEQ ID NO: 33 is the linker of the TNFRSF agonist fusion protein.

[0107] SEQ ID NO: 34 is the linker of the TNFRSF agonist fusion protein.

[0108] SEQ ID NO: 35 is the linker of the TNFRSF agonist fusion protein.

[0109] SEQ ID NO: 36 is the linker of the TNFRSF agonist fusion protein.

[0110] SEQ ID NO: 37 is the linker of the TNFRSF agonist fusion protein.

[0111] SEQ ID NO: 38 is the linker of the TNFRSF agonist fusion protein.

[0112] SEQ ID NO: 39 is the linker of the TNFRSF agonist fusion protein.

[0113] SEQ ID NO: 40 is the linker of the TNFRSF agonist fusion protein.

[0114] SEQ ID NO: 41 is the linker of the TNFRSF agonist fusion protein.

[0115] SEQ ID NO: 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0116] SEQ ID NO: 43 is the linker of the TNFRSF agonist fusion protein.

[0117] SEQ ID NO: 44 is the linker of the TNFRSF agonist fusion protein.

[0118] SEQ ID NO: 45 is the linker of the TNFRSF agonist fusion protein.

[0119] SEQ ID NO: 46 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0120] SEQ ID NO:47 is the soluble portion of the 4-1BBL polypeptide.

[0121] SEQ ID NO: 48 is the heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0122] SEQ ID NO: 49 is the light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0123] SEQ ID NO: 50 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0124] SEQ ID NO: 51 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0125] SEQ ID NO: 52 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E3-2.

[0126] SEQ ID NO: 53 is the light chain variable region (VL) of the 4-1BB agonist antibody H39E3-2.

[0127] SEQ ID NO: 54 is the amino acid sequence of human OX40.

[0128] SEQ ID NO: 55 is the amino acid sequence of mouse OX40.

[0129] SEQ ID NO: 56 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0130] SEQ ID NO: 57 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0131] SEQ ID NO: 58 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0132] SEQ ID NO: 59 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0133] SEQ ID NO: 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0134] SEQ ID NO: 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0135] SEQ ID NO: 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0136] SEQ ID NO: 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0137] SEQ ID NO: 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0138] SEQ ID NO: 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0139] SEQ ID NO: 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0140] SEQ ID NO: 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0141] SEQ ID NO: 68 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 11D4.

[0142] SEQ ID NO: 69 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 11D4.

[0143] SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0144] SEQ ID NO: 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0145] SEQ ID NO: 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0146] SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0147] SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0148] SEQ ID NO: 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0149] SEQ ID NO: 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0150] SEQ ID NO: 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0151] SEQ ID NO: 78 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 18D8.

[0152] SEQ ID NO: 79 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 18D8.

[0153] SEQ ID NO: 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0154] SEQ ID NO: 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0155] SEQ ID NO: 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0156] SEQ ID NO: 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0157] SEQ ID NO: 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0158] SEQ ID NO: 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0159] SEQ ID NO: 86 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu119-122.

[0160] SEQ ID NO: 87 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu119-122.

[0161] SEQ ID NO: 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0162] SEQ ID NO: 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0163] SEQ ID NO: 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0164] SEQ ID NO: 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0165] SEQ ID NO: 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0166] SEQ ID NO: 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0167] SEQ ID NO: 94 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu106-222.

[0168] SEQ ID NO: 95 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu106-222.

[0169] SEQ ID NO: 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0170] SEQ ID NO: 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0171] SEQ ID NO: 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0172] SEQ ID NO: 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0173] SEQ ID NO: 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0174] SEQ ID NO: 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0175] SEQ ID NO: 102 is the OX40 ligand (OX40L) amino acid sequence.

[0176] SEQ ID NO: 103 is the soluble portion of the OX40L polypeptide.

[0177] SEQ ID NO: 104 is an alternative soluble portion of the OX40L polypeptide.

[0178] SEQ ID NO: 105 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 008.

[0179] SEQ ID NO: 106 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 008.

[0180] SEQ ID NO: 107 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 011.

[0181] SEQ ID NO: 108 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 011.

[0182] SEQ ID NO: 109 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 021.

[0183] SEQ ID NO: 110 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 021.

[0184] SEQ ID NO: 111 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 023.

[0185] SEQ ID NO: 112 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 023.

[0186] SEQ ID NO: 113 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0187] SEQ ID NO: 114 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0188] SEQ ID NO: 115 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0189] SEQ ID NO: 116 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody.

[0190] SEQ ID NO: 117 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0191] SEQ ID NO: 118 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0192] SEQ ID NO: 119 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0193] SEQ ID NO: 120 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0194] SEQ ID NO: 121 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0195] SEQ ID NO: 122 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0196] SEQ ID NO: 123 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0197] SEQ ID NO: 124 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0198] SEQ ID NO: 125 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0199] SEQ ID NO: 126 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0200] SEQ ID NO: 127 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0201] SEQ ID NO: 128 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0202] SEQ ID NO: 129 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0203] SEQ ID NO: 130 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0204] SEQ ID NO: 131 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0205] SEQ ID NO: 132 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0206] SEQ ID NO: 133 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0207] SEQ ID NO: 134 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0208] SEQ ID NO: 135 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0209] SEQ ID NO: 136 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0210] SEQ ID NO: 137 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0211] SEQ ID NO: 138 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0212] SEQ ID NO: 139 is the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0213] SEQ ID NO: 140 represents the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0214] SEQ ID NO: 141 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0215] SEQ ID NO: 142 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0216] SEQ ID NO: 143 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0217] SEQ ID NO: 144 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0218] SEQ ID NO: 145 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0219] SEQ ID NO: 146 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0220] SEQ ID NO: 147 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0221] SEQ ID NO: 148 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0222] SEQ ID NO: 149 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0223] SEQ ID NO: 150 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0224] SEQ ID NO: 151 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0225] SEQ ID NO: 152 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0226] SEQ ID NO: 153 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0227] SEQ ID NO: 154 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0228] SEQ ID NO: 155 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0229] SEQ ID NO: 156 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0230] SEQ ID NO: 157 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0231] SEQ ID NO: 158 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0232] SEQ ID NO: 159 is the heavy chain variable region (V H ) amino acid sequence.

[0233] SEQ ID NO: 160 represents the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0234] SEQ ID NO: 161 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0235] SEQ ID NO: 162 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0236] SEQ ID NO: 163 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0237] SEQ ID NO: 164 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0238] SEQ ID NO: 165 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0239] SEQ ID NO: 166 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0240] SEQ ID NO: 167 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0241] SEQ ID NO: 168 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0242] SEQ ID NO: 169 is the heavy chain variable region (V H ) amino acid sequence.

[0243] SEQ ID NO: 170 represents the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0244] SEQ ID NO: 171 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0245] SEQ ID NO: 172 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0246] SEQ ID NO: 173 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0247] SEQ ID NO: 174 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0248] SEQ ID NO: 175 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0249] SEQ ID NO: 176 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0051] Detailed Description of the Invention

[0250] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0052] definition

[0251] The term "frozen" means that a tissue, cellular composition, or other composition has hardened or rigidified as a result of exposure to low temperatures. The term as used herein encompasses the concept of cryopreservation, which is the process of storing cells, tissues, etc. at very low temperatures that maintain the viability of the stored cells, tissues, etc.

[0053]

[0252] The term "cryostorage" refers to maintaining an already frozen composition at a very low temperature at which the stored cells, tissues, etc. maintain their viability.

[0054]

[0253] The terms "flash freezing," "snap freezing," "flash frozen," and "snap frozen" refer, by way of non-limiting example, to very rapid freezing in less than about 2 minutes.

[0055]

[0254] The terms "LOVO cell processing system" and "LOVO" refer to a cell processing system manufactured by Fresenius Kabi USA, LLC. These two terms also refer to any device or apparatus manufactured by any vendor that allows for continuous flow and cell processing in a sterile and / or closed environment, by pumping a cell-containing solution through a membrane or filter, such as a spinning membrane or spinning filter, and removing the supernatant or cell culture medium without pelleting. In some embodiments, such cell harvesters and / or cell processing systems can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0056]

[0255] 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 commercially available formulation of pimaricin or natamycin.

[0057]

[0256] The term "Fungizone" is a trademark of ERSquibb and Sons, LLC, and refers to the antifungal 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 commercially available formulation of amphotericin B.

[0058]

[0257] The term "physiologically buffered isotonic saline" refers to any one of many salt solutions known to those skilled in the art in which the solution is made at 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 can include Hank's balanced salt solution ("HBSS"), Tris-buffered saline ("TBS"), phosphate-buffered saline ("PBS"), or Dulbecco's phosphate-buffered saline ("DPBS" or "dPBS").

[0059]

[0258] The term "in vivo" refers to an event that takes place inside a subject's body.

[0060]

[0259] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, utilizing living or dead cells, and can also include cell-free assays, which do not utilize intact cells.

[0061]

[0260] The term "ex vivo" refers to events involving the treatment or implementation of treatment on cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body by way of surgery or treatment.

[0062]

[0261] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) over a one-week period, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a one-week period, or most preferably at least about 100-fold over a one-week period. Several rapid expansion protocols are outlined below.

[0063]

[0262] As used herein, "tumor-infiltrating lymphocytes" or "TILs" refer to a population of cells originally obtained as white blood cells that have left a subject's bloodstream 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 obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations expanded or grown as discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0064]

[0263] As used herein, a "cell population" (including TILs) refers to a large number of cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The range is 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8REP expansion cultures typically yield a bulk TIL population of 1.5 x 10 cells. 9 ~1.5×10 10 This is done to provide a population of cells for injection.

[0065]

[0264] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded culture (REP TILs), that are processed and stored at temperatures ranging from about -150°C to -60°C. General cryopreservation methods are also described elsewhere herein, including in the Examples. For clarity and to prevent misinterpretation, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs.

[0066]

[0265] As used herein, "thawed cryopreserved TILs" refers to a population of TILs that have been previously cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperatures or temperatures at which the TILs can be administered to a patient.

[0067]

[0266] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and affect treatment. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0068]

[0267] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 5% v / v DMSO to 10% v / v DMSO, and can also include media containing 7% v / v DMSO to 10% v / v DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium is sometimes referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium containing DMSO.

[0069]

[0268] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 hi ) and CD62L (CD62 hi ) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells predominate in the CD4 compartment of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0070]

[0269] The term "effector memory T cells" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+ but lack constitutive expression of CCR7 (CCR7lo) and have heterogeneous or low CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. After antigen stimulation, effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells have high amounts of perforin.

[0071]

[0270] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Examples of closed systems include, but are not limited to, closed G-containers. After tumor segments are added to the closed system, the system is not open to the external environment until immediately before the TILs are administered to a patient.

[0072]

[0271] The terms "fragmentation," "fragments," and "fragmented" as used herein to describe the process of disrupting tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method that disrupts the physical structure of tumor tissue.

[0073]

[0272] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[0074]

[0273] The term "anti-CD3 antibody" refers to an antibody or variant thereof, such as a monoclonal antibody, including human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0075]

[0274] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof, including a human, humanized, chimeric, or murine antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells, including OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or commercially available forms thereof, such as variants, conservative amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0076] [Table 1]

[0077] [Table 2]

[0078]

[0275] 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, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple sources at 22 million IU per single-use vial), as well as commercially available forms of recombinant IL-2 from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b), and other commercially available equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses pegylated forms of IL-2, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA, as described herein. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 2014 / 0328791 A1 and WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4902,502, the disclosures of which are incorporated herein by reference.Formulations of IL-2 suitable for use in the present invention are described in US Pat. No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0079]

[0276] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells as well as by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell expansion and class II MHC expression and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0080]

[0277] The term "IL-7" (also referred to herein as "IL7") refers to the glycosylated tissue-derived cytokine known as interleukin-7, which is available from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, in a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0081]

[0278] 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, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0082]

[0279] 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, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc., 13:379-95 (2014), the disclosure of which is incorporated herein by reference. IL-21 is produced primarily 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 weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0083]

[0280] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including but not limited to disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro, in vivo, and ex vivo), the subject and disease state being treated (e.g., the subject's weight, age, and sex), the severity of the disease state, or the method of administration, among other factors, which can be readily determined by one of ordinary skill in the art. The term also applies to a dose that elicits a specific response in target cells (e.g., decreased platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0084]

[0281] A "therapeutic effect," as that term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit. A prophylactic benefit 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.

[0085]

[0282] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0086]

[0283] When ranges are used herein to describe physical or chemical properties, such as, for example, molecular weight or chemical formula, all combinations and subcombinations of ranges, as well as specific embodiments therein, are intended to be included. The use of the term "about" when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is an approximation within experimental variation (or within statistical experimental error), and thus the numerical value or numerical range may vary. The variation is typically 0% to 15%, preferably 0% to 10%, and more preferably 0% to 5% of the stated numerical value or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes embodiments, such as, for example, embodiments of any composition of matter, method, or process that "consists of" or "consists essentially of" the described property.

[0087]

[0284] The compounds of the present 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. "Antibody" also 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 comprises a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which can be interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen epitopes. The constant region of the antibody can 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.

[0088]

[0285] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody (e.g., IL-33, ST2, CD20, PD-1, PD-L1, or PD-L2) that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L(ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or V L and (vi) isolated complementarity-determining regions (CDRs). In addition, two domains of an Fv fragment, V, and VB, may be included. L and V H are encoded by separate genes, which can be synthesized by recombinant methods. L and V H Pairs of domains can be linked by a synthetic linker, allowing them to be produced as a single protein chain forming a monovalent molecule known as a single-chain Fv (scFv); see, e.g., Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0089]

[0286] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0090]

[0287] 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, human monoclonal antibodies are produced by hybridomas obtained from transgenic non-human animals, e.g., transgenic mice, whose genomes include human heavy chain and light chain transgenes fused to an immortalized cell.

[0091]

[0288] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express human antibodies; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means, including 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. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V and V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is similar to that of the human germline V H and V L These are sequences that, while derived from and related to sequences, may not naturally occur within the human antibody germline repertoire in vivo.

[0092]

[0289] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. Five antibody isotypes exist in mammals: IgA, IgD, IgG, IgM, and IgE. In humans, the IgG isotype has four subclasses: IgG1, IgG2, IgG3, and IgG4, and the IgA isotype has two subclasses: IgA1 and IgA2.

[0093]

[0290] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0094]

[0291] The term "human antibody derivative" refers to any mutant form of a human antibody, for example, a conjugate of an antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to a therapeutic moiety, such as a bacterial toxin, a cytotoxic drug, or a radionuclide-containing toxin. Toxic moieties can be conjugated to the antibodies of the invention using methods available in the art.

[0095]

[0292] The terms "humanized antibody" or "humanized" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's hypervariable region have been replaced by residues from 15 hypervariable regions of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin have been replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, 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.

[0096]

[0293] 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 antibodies in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0097]

[0294] A "diabody" is a small antibody fragment with two antigen-binding sites. The fragments are bound to the same polypeptide chain (V H -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Diabodies are further described in, for example, EP 404,097; WO 93 / 11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0098]

[0295] The term "glycosylation" refers to an engineered derivative of an antibody. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered glycosylation types, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha(1,6) fucosyltransferase), and therefore antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. Ms704, Ms705, and Ms709 FUT8 - / -The cell line was generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Application Publication No. 2004 / 0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent No. 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. The document also describes cell lines, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662), with reduced or no enzymatic activity that adds fucose to N-acetylglucosamine attached to the Fc region of antibodies. WO 03 / 035835 describes a mutant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates and results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol Chem. 2002, 277, 26733-26740). WO 99 / 54342 describes cell lines that have been engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (Umana, et al., Nat. Biotech. 1999, 17, (See also 176-180.) Alternatively, the fucose residues of antibodies can be cleaved 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.

[0099]

[0296] "PEGylation" refers to a modified antibody or fragment thereof that is reacted with polyethylene glycol (PEG), typically a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 The term "PEG" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods of pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. 0154316 and 0401384.

[0100]

[0297] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference anti-CD20 monoclonal antibody is rituximab, an anti-CD20 biosimilar monoclonal antibody approved by a drug regulatory agency for rituximab is a "biosimilar to" or "biosimilar of" rituximab. In Europe, a similar biological or "biosimilar" medicinal product is one that is similar to another biological product already authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, a biosimilar may be authorized, granted authorization, or the subject of an authorization application under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. In Europe, the original biological product already authorized is sometimes referred to as the "reference medicinal product." Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guideline on Similar Biological Medicinal Products. Additionally, product-specific guidelines, including those for monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are published on its website.Biosimilars described herein may be similar to the reference medicinal product in terms of quality attributes, biological activity, mechanism of action, safety profile, and / or efficacy. Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality attributes to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively, or in addition, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to reference medicinal products authorized by the EMA. However, in some cases, biosimilars may be compared to biological medicinal products authorized outside the European Economic Area (non-EEA "comparators") in specific studies. Such studies include, for example, specific clinical trials and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to a biological drug that has been or can be compared to a non-EEA-approved comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may include an amino acid sequence with 97% or more sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference drug. A biosimilar may contain one or more post-translational modifications that differ from those of the reference drug, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, provided that the differences do not result in changes in the safety and / or efficacy of the drug. A biosimilar may have the same or a different glycosylation pattern as the reference drug.Biosimilars may have different glycosylation patterns, particularly, but not exclusively, if the differences address or are intended to address safety concerns related to the reference drug. Furthermore, biosimilars may deviate from the reference drug, for example, in its strength, pharmaceutical form, formulation, excipients, and / or presentation, provided that the drug's safety and efficacy are not compromised. Biosimilars may contain, for example, differences in pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles compared to the reference drug, but still be deemed sufficiently similar to the reference drug to be approved or considered suitable for approval. In certain circumstances, biosimilars exhibit different binding properties compared to the reference drug, where the different binding properties are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0101]

[0298] The term "hematologic malignancies" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematologic malignancies are also referred to as "liquid tumors." Hematologic malignancies include, but are not limited to, ALL, CLL, SLL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B cells.

[0102]

[0299] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells), and supporting stromal cells in which cancer cells may disperse and provide a supportive microenvironment.

[0103]

[0300] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors are also referred to herein as bone marrow-infiltrating lymphocytes (MILs).

[0104]

[0301] The term "microenvironment" as used herein may refer to the solid or hematologic tumor microenvironment as a whole or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to the complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, cultivate therapeutic resistance, and provide a niche for successful and dominant metastasis, as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that are recognized by T cells, elimination of tumors by the immune system is rare due to immunosuppression by the microenvironment.

[0105]

[0302] In certain embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In some embodiments, a TIL population can be provided, wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In one embodiment, the non-myeloablative chemotherapy comprises cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m for 5 days (27-23 days before TIL infusion). 2 In one embodiment, after non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), the patient receives an intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours to a physiologically tolerated dose.

[0106]

[0303] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") on patients prior to introducing the rTILs of the present invention.

[0107]

[0304] The terms "sequence identity," "percent identity," and "percent sequence 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 the same nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine appropriate parameters for maximal alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0108]

[0305] Certain embodiments of the present invention include variants of antibodies, such as anti-IL-33 or anti-ST2 antibodies and / or anti-CD20 antibodies and / or anti-PD-1 antibodies, anti-PD-L1 and / or anti-PD-L2 antibodies. As used herein, the term "variant" encompasses, but is not limited to, antibodies containing an amino acid sequence that differs from that of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to the antigen.

[0109]

[0306] For the avoidance of doubt, it is intended that a particular feature (e.g., an integer, property, value, use, disease, formula, compound, or group) described herein in connection with a particular aspect, embodiment, or example of the invention is to be understood as also applicable to any other aspect, embodiment, or example described herein, unless incompatible. Accordingly, such features may be used, where appropriate, with any of the definitions, claims, or embodiments defined herein. All features disclosed herein (including the accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except where combinations of at least some features and / or steps are mutually exclusive. The invention is not limited to any of the details of the disclosed embodiments. The invention extends to any novel or novel combination of features disclosed herein (including the accompanying claims, abstract, and drawings), or any novel or novel combination of steps of any method or process so disclosed.

[0110] Tumor cryopreservation method

[0307] The present invention provides (i) Fragmenting tumor tissue; (ii) incubating the fragments in cryopreservation medium; and (iii) Freezing the fragments, the freezing being flash freezing using the vapor phase of liquid nitrogen. The present invention provides a method for cryopreserving tumor tissue for the production of tumor infiltrating lymphocytes (TILs), comprising:

[0111]

[0308] In one 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 one embodiment, the approximately spherical fragments have a diameter of about 3 mm.

[0112]

[0309] In some embodiments, the tumor tissue is fragmented into approximately rectangular pieces having a shortest side length of at least 1.5 mm and a longest side length of about 6 mm. In one embodiment, the tumor tissue is fragmented into approximately cubic pieces having a side length of about 1.5 mm to 6 mm. In one embodiment, the approximately cubic pieces have a side length of about 6 mm. In one embodiment, the approximately cubic pieces have a side length of about 3 mm.

[0113]

[0310] In some embodiments, the tissue sample is trimmed to separate non-tumor tissue from tumor tissue.

[0114]

[0311] In some embodiments, the tumor tissue is from a dissected tumor. In one 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 excision biopsy. In some embodiments, the tumor tissue may be from one or more core needle biopsies.

[0115]

[0312] In some embodiments, fresh tumor tissue is trimmed into pieces with cross-sections of about 1.5 mm x 1.5 mm, about 2 mm x 2 mm, about 2.5 mm x 2.5 mm, about 3 mm x 3 mm, about 3.5 mm x 3.5 mm, about 4 mm x 4 mm, about 4.5 mm x 4.5 mm, about 5 mm x 5 mm, about 5.5 mm x about 5.5 mm, or about 6 mm x about 6 mm.

[0116]

[0313] In one embodiment, the tumor tissue is less than 12 hours old. In one embodiment, the tumor tissue is less than 8 hours old. In one embodiment, the tumor sample is less than 3 hours, less than 2 hours, or less than 1 hour old.

[0117]

[0314] In some embodiments, the cryopreservation medium comprises 2-12% v / v (volume:volume) dimethyl sulfoxide (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 5-10% 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.

[0118]

[0315] In some embodiments, the cryopreservation medium comprises 2-12% w / w (weight:weight) dimethyl sulfoxide (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 5-10% 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.

[0119]

[0316] In one embodiment, the cryopreservation medium comprises at least one antibacterial agent. In one embodiment, the at least one antibacterial agent is gentamicin at a concentration of at least 50 μg / mL. In some embodiments, the at least one antibacterial agent is penicillin; in some embodiments, the at least one antibacterial agent is streptomycin. In some embodiments, the at least one antibacterial agent is an antifungal agent. In some embodiments, the antifungal agent is amphotericin B; in some embodiments, the antifungal agent is Fungin™. In some embodiments, a combination of antibacterial agents is used. In some embodiments, at least one antifungal agent is used in combination with one or more antibacterial agents.

[0120]

[0317] In some embodiments, the tumor fragments are incubated in the cryopreservation medium for about 20 minutes to about 70 minutes. In some embodiments, the tumor fragments are incubated in the cryopreservation medium for about 30 minutes to about 60 minutes. In some embodiments, the incubation is for 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 for 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 for 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 the tumor fragment density, hi some embodiments, the incubation time is proportional to the surface-to-volume ratio of the tumor fragments.

[0121]

[0318] In some embodiments, the tumor fragments are incubated in cryopreservation medium at a temperature of about 2°C to about 8°C.

[0122]

[0319] In some embodiments, the tumor tissue is washed with physiologically buffered isotonic saline. In some embodiments, the washing comprises three successive washes of at least 3 minutes each, with the physiologically buffered isotonic saline being replaced after each successive wash. In some embodiments, the physiologically buffered isotonic saline comprises Hank's Balanced Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline comprises Tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline comprises phosphate-buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline comprises Dulbecco's Phosphate-buffered Saline (DPBS). In some embodiments, the physiologically buffered isotonic saline in one successive wash may be a different physiologically buffered isotonic saline from that used in one or more other successive washes.

[0123]

[0320] In one embodiment, freezing occurs at a temperature ranging from about −125° C. to about −196° C. In one embodiment, freezing occurs at a temperature ranging from about −140° C. to about −185° C. In one embodiment, freezing occurs at a temperature ranging from about −140° C. to about −175° C. In one embodiment, freezing occurs at a temperature of about −145° C. In some embodiments, freezing occurs in the vapor phase of liquid nitrogen.

[0124]

[0321] A well-known problem in the art is cryopreserving cells or tissues without damaging them during the freezing process. Without being bound by theory, one cause of damage during freezing is intracellular ice nucleation, which causes cells to rupture. Muldrew and McGann, in "The osmotic rupture hypothesis of intracellular freezing injury," Biophysical Journal, 66:532-41 (1994), outlined a quantitative theory of this well-known and widely recognized difficulty with cryopreservation of cells, particularly whole tissues. Acker and McGann further developed the basic mechanisms of intracellular ice formation and cell damage in a later paper, "Membrane damage occurs during the formation of intracellular ice," Cryo Letter, 22:241-54 (2001).

[0125]

[0322] Damage during freezing is detected when the tissue substantially loses its physiological structure or the cells comprising the tissue substantially lose their viability upon thawing. Viability can be determined by the percentage of cells introduced into the culture medium compared to the number of cells that proliferate or exhibit markers of normal cell function. Many methods for identifying the fraction of viable cells are known in the art, including, but not limited to, 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. Viability can also be determined by metabolic activity assays, such as, but not limited to, the MTT assay, in which MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, is metabolized to formazan by cellular enzymes. This enzymatic reaction converts the yellow MTT to purple formazan. See, e.g., 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).

[0126]

[0323] Without being bound by theory, it is hypothesized that slow cooling produces harmless intracellular ice. See Acker and McGann, "Protective effect of intracellular ice during freezing?" Cryobiology, 46(2):197-202 (2003). A traditional approach to achieving freezing without excessive cell damage or significantly reducing cell viability has been to employ a slow freezing rate. U.S. Pat. No. 5,891,617 (Watson et al.) emphasizes this approach; for example, step (c) of claim 1 teaches a very slow cooling rate: "about -0.3°C per minute or less." Similarly, U.S. Pat. No. 9,938,495 (Comhaire et al.) teaches that "slow freezing using a DMSO-free cryopreservation medium resulted in high post-thaw cell viability" of stem cells.

[0127]

[0324] Based on these exemplary teachings, the methods and products of the present disclosure are surprising and unexpected. Moreover, the present disclosure, including the examples and data therein, demonstrates a technical solution to the problem of rapidly and efficiently cryoprotecting tumor tissue, tumor fragments, or tumor specimens for use in producing tumor-infiltrating lymphocytes for therapeutic use.

[0128]

[0325] In some embodiments, the method for cryopreserving tumor tissue for the production of tumor infiltrating lymphocytes (TILs) further comprises step (iv) storing the frozen fragments at a temperature of at least less than −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 in liquid nitrogen. In some embodiments, the cryopreserved fragments are stored for later production of TILs for autologous therapeutic applications.

[0129]

[0326] The present invention provides (a) storing tumor tissue in a frozen state, the method of storing tumor tissue comprising incubating the tumor tissue in a storage medium at a temperature of about 2°C to about 8°C for about 20 minutes to about 70 minutes prior to freezing; (b) thawing the tumor tissue; (c) treating the tumor tissue in the gas-permeable container with a first cell culture medium containing interleukin-2 (IL-2) and optionally an OKT-3 antibody to provide TILs; (d) removing at least a plurality of TILs; and (e) expanding the TILs in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0130]

[0327] The present invention provides (a) fragmenting a tumor sample; (b) incubating the tumor fragments in storage medium; (c) storing the tumor fragments in a frozen state; (d) thawing the tumor fragments; (e) treating the tumor fragments in the gas-permeable container with a first cell culture medium containing interleukin-2 (IL-2) and optionally an OKT-3 antibody to provide TILs; (f) removing at least a plurality of TILs; and (g) expanding the TILs in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0131]

[0328] In some embodiments, the storage medium comprises 2-12% v / v (volume:volume or volume to volume) dimethyl sulfoxide (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 5-10% 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.

[0132]

[0329] In some embodiments, the storage medium comprises 2-12% w / w (weight:weight or weight to weight) dimethyl sulfoxide (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 5-10% 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.

[0133]

[0330] In some embodiments, the tumor tissue is incubated in storage medium at a temperature of about 2°C to about 8°C for about 20 minutes to about 70 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature of about 2°C to about 8°C for at least 30 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature of about 2°C to about 8°C for 30 minutes to about 60 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 15 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 45 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 60 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2° C. to about 8° C. for about 75 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2° C. to about 8° C. for about 60 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2° C. to about 8° C. for 60 minutes before freezing.

[0134]

[0331] In some embodiments, the storage medium comprises about 5% v / v DMSO, and the tumor tissue is incubated in the storage medium at a temperature of about 2°C to about 8°C for about 60 minutes before freezing. In some embodiments, the storage medium comprises about 10% v / v DMSO, and the tumor tissue is incubated in the storage medium at a temperature ranging from about 2°C to about 8°C for at least 30 minutes to about 60 minutes before freezing. In some embodiments, the incubation time is proportional to the tumor fragment density. In some embodiments, the incubation time is proportional to the surface-to-volume ratio of the tumor fragments.

[0135] Cryopreserved tumor fragments

[0332] In some embodiments, the present invention provides a method for treating a skin condition comprising: (i) fragmenting the tumor sample; (ii) incubating the fragments in cryopreservation medium; and (iii) freezing the fragments, the freezing being flash freezing using the vapor phase of liquid nitrogen; The present invention provides cryopreserved tumor fragments for the production of tumor-infiltrating lymphocytes (TILs), prepared by a process comprising:

[0136]

[0333] In some embodiments, the present invention provides a method for treating a skin condition comprising: (i) fragmenting the tumor sample; (ii) incubating the fragments in a cryopreservation medium containing 10% v / v DMSO at a temperature ranging from about 2°C to about 8°C for about 20 minutes to about 70 minutes; and (iii) freezing the fragments, the freezing being flash freezing using the vapor phase of liquid nitrogen; The present invention provides cryopreserved tumor fragments for the production of tumor-infiltrating lymphocytes (TILs), prepared by a process comprising:

[0137]

[0334] In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 6 mm. In one embodiment, the approximately spherical fragments have a diameter of about 6 mm. In one embodiment, the approximately spherical fragments have a diameter of about 3 mm. In one 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.

[0138]

[0335] In some embodiments, the tumor sample is fragmented into approximately rectangular pieces having a shortest side length of at least 1.5 mm and a longest side length of about 6 mm. In one embodiment, the tumor sample is fragmented into approximately cubic pieces having sides between about 1.5 mm and 6 mm long. In one embodiment, the approximately cubic pieces have sides that are about 6 mm long. In one embodiment, the approximately cubic pieces have sides that are about 3 mm long. In one embodiment, the approximately cubic pieces have a side 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.

[0139]

[0336] In one embodiment, the tumor sample is about 2 mm 3 ~about 200mm 3 In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 5 mm 3 ~about 150mm 3 In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 25 mm 3 ~about 150mm 3 In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 50 mm. 3 ~about 150mm 3 In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 100 mm 3 ~approx. 125mm 3 In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 50 mm. 3 ~about 75mm 3 In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 75 mm 3 ~approx. 100mm 3 In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 200 mm 3 ~approx. 1000mm 3In one embodiment, the tumor sample is fragmented into approximately spherical fragments having a volume of about 500 mm 3 ~approx. 800mm 3 In one embodiment, the substantially spherical pieces 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.

[0140]

[0337] In some embodiments, fresh tumor tissue is trimmed into pieces, wherein the pieces have a cross-section of about 1.5 mm x 1.5 mm, about 2 mm x 2 mm, about 2.5 mm x 2.5 mm, about 3 mm x 3 mm, about 3.5 mm x 3.5 mm, about 4 mm x 4 mm, about 4.5 mm x 4.5 mm, about 5 mm x 5 mm, about 5.5 mm x about 5.5 mm, about 6 mm x 6 mm, about 6.5 mm x 6.5 mm, about 7 mm x 7 mm, about 7.5 mm x 7.5 mm, about 8 mm x 8 mm, about 8.5 mm x 8.5 mm, about 9 mm x 9 mm, about 9.5 mm x 9.5 mm, about 10 mm x 10 mm, about 10.5 mm x 10.5 mm, about 11 mm x 11 mm, about 11.5 mm x 11.5 mm, about 12 mm x 12 mm.

[0141]

[0338] In some embodiments, the fresh tumor tissue is trimmed into pieces, wherein the pieces are about 2 mm 2 ~about 3mm 2 , about 3mm 2 ~about 4mm 2 , about 4mm 2 ~about 5mm 2 , about 5mm 2 ~about 6mm 2 , about 6mm 2 ~about 7mm 2 , about 7mm 2 ~approx. 8mm 2 , approximately 8 mm 2 ~approx. 9mm 2 , approximately 9 mm 2 ~about 10mm 2 , about 10mm 2 ~approx. 11mm2 , about 11mm 2 ~approx. 12mm 2 , approximately 12 mm 2 ~about 20mm 2 , about 20mm 2 ~approx. 50mm 2 , approximately 50 mm 2 ~approx. 100mm 2 , or about 100 mm 2 ~approx. 500mm 2 In some embodiments, the fresh tumor tissue is trimmed into pieces, wherein the pieces have a cross-sectional area of ​​about 1.5 mm 2 , about 2 mm 2 , about 2.5mm 2 , about 3mm 2 , about 3.5mm 2 , about 4mm 2 , about 4.5mm 2 , about 5mm 2 , about 5.5mm 2 , about 6mm 2 , about 6.5mm 2 , about 7mm 2 , about 7.5mm 2 , approximately 8 mm 2 , about 8.5mm 2 , approximately 9 mm 2 , about 9.5mm 2 , about 10mm 2 , about 10.5mm 2 , about 11mm 2 , about 11.5mm 2 , approximately 12 mm 2 , about 20mm 2 , about 25mm 2 , about 30mm 2 , approximately 40 mm 2 , approximately 50 mm 2 , approximately 100 mm 2 , about 200mm 2 , about 300mm 2 , approximately 400 mm 2 , approximately 500 mm 2 , about 750mm 2 , or approximately 1000 mm 2 has a cross-sectional area of

[0142]

[0339] In some embodiments, the fresh tumor tissue is trimmed into pieces, wherein the pieces are about 2 mm 3 ~about 3mm 3 , about 3mm 3 ~about 4mm 3 , about 4mm 3 ~about 5mm 3 , about 5mm 3 ~about 6mm 3 , about 6mm 3 ~about 7mm 3 , about 7mm 3 ~approx. 8mm 3 , approximately 8 mm 3 ~approx. 9mm 3 , approximately 9 mm 3 ~about 10mm 3 , about 10mm 3 ~approx. 11mm 3 , about 11mm 3 ~approx. 12mm 3 , and approximately 12 mm 3 ~about 20mm 3 In some embodiments, the fresh tumor tissue is trimmed into pieces, wherein the pieces have a volume of about 1.5 mm 3 , about 2 mm 3 , about 2.5mm 3 , about 3mm 3 , about 3.5mm 3 , about 4mm 3 , about 4.5mm 3 , about 5mm 3 , about 5.5mm 3 , about 6mm 3 , about 6.5mm 3 , about 7mm 3 , about 7.5mm 3 , approximately 8 mm 3 , about 8.5mm 3 , approximately 9 mm 3 , about 9.5mm 3 , about 10mm 3 , about 10.5mm 3 , about 11mm 3 , about 11.5mm 3 , approximately 12 mm 3 , about 20mm 3 , about 25mm 3 , about 30mm 3 , approximately 40 mm 3 , approximately 50 mm3 , approximately 100 mm 3 , about 200mm 3 , about 300mm 3 , approximately 400 mm 3 , approximately 500 mm 3 , about 750mm 3 , or approximately 1000 mm 3 It has a volume of

[0143]

[0340] In some embodiments, the tumor sample is from a dissected tumor. In one 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 excision biopsy. In some embodiments, the tumor sample may be from one or more core needle biopsies.

[0144]

[0341] In one embodiment, the tumor sample is less than 12 hours before freezing. In one embodiment, the tumor sample is less than 8 hours before freezing. In one embodiment, the tumor sample is less than 3 hours, less than 2 hours, or less than 1 hour before freezing.

[0145]

[0342] In some embodiments, the cryopreservation medium comprises 2-12% v / v (volume:volume or volume to volume) dimethyl sulfoxide (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 5-10% 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.

[0146]

[0343] In some embodiments, the cryopreservation medium comprises 2-12% w / w (weight:weight or weight to weight) dimethyl sulfoxide (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 5-10% 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.

[0147]

[0344] In one embodiment, the cryopreservation medium comprises at least one antimicrobial agent. In one 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 antimicrobial agent is penicillin; in some embodiments, the at least one antimicrobial 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 Fungin™. In some embodiments, a combination of antimicrobial agents is used. In some embodiments, at least one antifungal agent is used in combination with one or more antimicrobial agents.

[0148]

[0345] In some embodiments, the tumor fragments are incubated in the cryopreservation medium for about 30 minutes to about 60 minutes. In some embodiments, the incubation is for 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 the tumor fragment density. In some embodiments, the incubation time is proportional to the surface-to-volume ratio of the tumor fragments.

[0149]

[0346] In some embodiments, the tumor fragments are incubated in cryopreservation medium at a temperature of about 2°C to about 8°C.

[0150]

[0347] In some embodiments, the tumor sample is washed with physiologically buffered isotonic saline. In some embodiments, the washing comprises three successive washes of at least 3 minutes each, with the physiologically buffered isotonic saline being replaced after each successive wash. In some embodiments, the physiologically buffered isotonic saline comprises Hank's Balanced Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline comprises Tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline comprises phosphate-buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline comprises Dulbecco's Phosphate-buffered Saline (DPBS). In some embodiments, the physiologically buffered isotonic saline in one successive wash may be a different physiologically buffered isotonic saline than that used in one or more other successive washes.

[0151]

[0348] In one embodiment, freezing occurs at a temperature ranging from about −125° C. to about −180° C. In one embodiment, freezing occurs at a temperature ranging from about −140° C. to about −175° C. In one embodiment, freezing occurs at a temperature of about −145° C. In some embodiments, freezing occurs in the vapor phase of liquid nitrogen.

[0152]

[0349] In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is one. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is two. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is three. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is four. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is five. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is six. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is seven. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is eight. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is nine. In one embodiment, the number of tumor fragments used in accordance with any of the embodiments above or herein is ten.

[0153] Production of TILs from cryopreserved tumor fragments

[0350] The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from cryopreserved tumor fragments into a therapeutic TIL population, the method comprising: (i) obtaining a first population of TILs from a cryopreserved tumor fragment; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, and optionally OKT-3, to generate a second TIL population; and (iii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold or 100-fold more numerous than the second TIL population, and the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population comprising increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population. Includes:

[0154]

[0351] The present invention provides a method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy, the method comprising: (a) culturing cryopreserved tumor fragments in a first cell culture medium containing interleukin 2 (IL-2) and optionally an OKT-3 antibody; (b) expanding the TILs in a second cell culture medium containing 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; Includes:

[0155]

[0352] The present invention provides (a) storing tumor tissue in a frozen state, the method of storing tumor tissue comprising incubating the tumor tissue in a storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes prior to freezing; (b) thawing the tumor tissue; (c) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (d) removing at least a plurality of TILs; and (e) expanding the TILs in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0156]

[0353] The present invention provides (a) fragmenting a tumor sample; (b) incubating the tumor fragments in the storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (c) storing the tumor fragments in a frozen state; (d) thawing the tumor fragments; (e) treating the tumor fragments in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (f) removing at least a plurality of TILs; and (g) expanding the TILs in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0157]

[0354] In some embodiments, the storage medium comprises 2% v / v DMSO to 12% v / v dimethyl sulfoxide (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 about 5% v / v DMSO to 10% v / v DMSO.

[0158]

[0355] In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for at least 30 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for 30 minutes to about 60 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 45 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 60 minutes before freezing. In some embodiments, the tumor tissue is incubated in storage medium at a temperature ranging from about 2°C to about 8°C for about 60 minutes before freezing.

[0159]

[0356] In some embodiments, the tumor tissue is incubated in the cryopreservation medium for about 20 minutes to about 70 minutes. In some embodiments, the tumor tissue is incubated in the cryopreservation medium for about 30 minutes to about 60 minutes. In some embodiments, the incubation is for 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 the tumor fragment density. In some embodiments, the incubation time is proportional to the surface-to-volume ratio of the tumor fragments.

[0160]

[0357] In some embodiments, tumor fragments measuring approximately 1.5 mm x 1.5 mm in cross section are incubated in cryopreservation medium for less than about 20 minutes, about 30 minutes, about 35 minutes, about 40 minutes, or about 60 minutes. In some embodiments, tumor fragments measuring approximately 2 mm x 2 mm in cross section are incubated in cryopreservation medium for about 20 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, or about 60 minutes. In some embodiments, tumor fragments measuring approximately 3 mm x 3 mm in cross section are incubated in cryopreservation medium for at least 20 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, or about 60 minutes. In some embodiments, tumor fragments measuring approximately 4 mm x 4 mm in cross section are incubated in cryopreservation medium for about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 60 minutes, or about 70 minutes. In some embodiments, tumor fragments measuring about 5 mm x 5 mm in cross section are incubated in cryopreservation medium for about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 60 minutes, or about 70 minutes. In some embodiments, tumor fragments measuring about 6 mm x 6 mm in cross section are incubated in cryopreservation medium for at least 20 minutes, about 30 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 60 minutes, or about 70 minutes.

[0161]

[0358] In some embodiments, the storage medium comprises about 5% v / v DMSO and the tumor tissue is incubated in the storage medium for about 60 minutes prior to freezing at a temperature ranging from about 2° C. to about 8° C. In some embodiments, the storage medium comprises about 10% v / v DMSO and the tumor tissue is incubated in the storage medium for at least about 30 minutes to about 60 minutes prior to freezing at a temperature ranging from about 2° C. to about 8° C.

[0162]

[0359] In some embodiments, a method for expanding TILs from frozen tumor tissue comprises incubating cryopreserved tumor fragments in a culture medium containing IL-2, wherein the number of TILs is 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 more than 2500-fold.

[0163]

[0360] In some embodiments, the method for producing TILs from frozen tumor tissue comprises: (a) storing tumor tissue in a frozen state, the method of storing tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 minutes to about 70 minutes; (iv) refrigerating the container; and (v) storing the container so that it remains frozen; including; (b) thawing the container; (c) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (d) removing at least a plurality of TILs; and (e) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas-permeable container to provide an expanded number of TILs. Includes:

[0164]

[0361] In some embodiments, the method for producing TILs from frozen tumor tissue comprises: (a) Obtaining tumor tissue from the patient; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable container containing a storage medium; (d) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 minutes to about 70 minutes; (e) Freezing the container; (f) storing the container so that it remains frozen; (g) thawing the container; (h) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (i) removing at least a plurality of TILs; and (j) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas-permeable container to provide an expanded number of TILs. Includes:

[0165]

[0362] In some embodiments, the method of producing TILs from cryopreserved tumor fragments further comprises adding OKT-3 to the first culture medium.

[0166]

[0363] In some embodiments, the fresh tumor tissue is trimmed to a diameter of 1.5 mm to 6 mm and a thickness of 1.5 mm to 6 mm. In yet other embodiments, the fresh tumor tissue is trimmed to approximately 6 mm x 6 mm x 6 mm. In some embodiments, the fresh tumor tissue is trimmed to pieces with cross-sections of approximately 1.5 mm x 1.5 mm, approximately 2 mm x 2 mm, approximately 2.5 mm x 2.5 mm, approximately 3 mm x 3 mm, approximately 3.5 mm x 3.5 mm, approximately 4 mm x 4 mm, approximately 4.5 mm x 4.5 mm, approximately 5 mm x 5 mm, approximately 5.5 mm x approximately 5.5 mm, or approximately 6 mm x 6 mm.

[0167]

[0364] 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 μg / mL to about 50 μg / mL.

[0168]

[0365] In some embodiments, the tumor tissue is washed with physiologically buffered isotonic saline. In some embodiments, the washing comprises three successive washes of at least 3 minutes each, with the physiologically buffered isotonic saline being replaced after each successive wash. In some embodiments, the physiologically buffered isotonic saline comprises Hank's Balanced Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline comprises Tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline comprises phosphate-buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline comprises Dulbecco's Phosphate-buffered Saline (DPBS). In some embodiments, the physiologically buffered isotonic saline in one successive wash may be a different physiologically buffered isotonic saline from that used in one or more other successive washes.

[0169]

[0366] In some embodiments, the thawing step comprises immersing the container in a 37° C. water bath for about 5 minutes.

[0170]

[0367] In some embodiments, the freezing step comprises freezing the container at about −125° C. to about −195° C. In some embodiments, the container is frozen at a temperature of about −125° C. to about −150° C.; in some embodiments, the container is frozen at a temperature of about −125° C. to about −145° C.; in further embodiments, the container is frozen at a temperature of about −135° C.

[0171]

[0368] In some embodiments, the method is performed using a fresh tumor sample from a human subject.

[0172]

[0369] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney 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.

[0173]

[0370] In some embodiments, the method further comprises splitting between the first expansion culture (or first culture) step and the second expansion culture (or second culture) step. In one embodiment, the splitting occurs on about day 16. In some embodiments, the first expansion culture (or first culture) step is completed in about 11 days. In other embodiments, the first expansion culture (or first culture) step is completed in about 7 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 7 days, and the second expansion culture (or second culture) step is completed in about 14 days. In some embodiments, steps (c) to (e) are completed in about 22 days. In still other embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are completed in about 21 days; in some embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are completed in about 20 days; in some embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are completed in about 16 days.

[0174]

[0371] In some embodiments, the first culture medium comprises OKT-3.

[0175]

[0372] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) storing tumor tissue in a frozen state, the method of storing tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (iv) freezing the container using the vapor phase of liquid nitrogen; and (v) Storing the container in the vapor phase at liquid nitrogen temperature. including; (b) thawing the tumor tissue; (c) adding tumor tissue to a closed system; (d) performing a first expansion culture by culturing a first TIL population from the tumor tissue in a cell culture medium having IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed container providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system; (f) recovering the therapeutic TIL population obtained from step (f), wherein the transition from step (e) to step (f) occurs without releasing the system; (g) transferring the TIL population collected in step (f) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag containing the TIL population collected in step (g) using a cryopreservation process; and (i) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the subject. Includes:

[0176]

[0373] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) obtaining tumor tissue from a subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable container containing a storage medium; (d) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (e) freezing the container using the vapor phase of liquid nitrogen; (f) storing the container in the vapor phase at liquid nitrogen temperatures; (g) thawing the tumor tissue; (h) adding tumor tissue to a closed system; (i) conducting a first expansion culture in a closed system by culturing a first TIL population from the tumor tissue in a cell culture medium having IL-2 to generate a second TIL population, wherein the first expansion culture is conducted in a closed vessel providing a first gas permeable surface area, the first expansion culture is conducted for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (i) to step (j) occurs without opening the system; (k) recovering the therapeutic TIL population obtained from step (j), wherein the transition from step (j) to step (k) occurs without releasing the system; (l) transferring the therapeutic TILs collected in step (k) to an infusion bag, wherein the transition from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag containing the therapeutic TIL population from step (l) using a cryopreservation process; (n) thawing the therapeutic TIL population in the infusion bag from step (m); and (o) administering a therapeutically effective dose of the therapeutic TIL population to the subject from the infusion bag in step (n). The method includes administering expanded tumor-infiltrating lymphocytes (TILs), comprising:

[0177]

[0374] 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, resistant melanoma, metastatic melanoma, and non-small cell lung cancer. The histology of solid tumors comprises interdependent tissue compartments, including parenchyma (cancer cells), and supporting stromal cells in which cancer cells may disperse and provide a supportive microenvironment.

[0178]

[0375] The present invention provides (a) obtaining tumor tissue from a patient, the tumor tissue comprising TILs; (b) storing the tumor tissue in a frozen state, wherein the method of storing the tumor tissue comprises incubating the tumor tissue in storage medium at 2°C to 8°C for 30 minutes prior to freezing; (c) thawing the tumor tissue; (d) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (e) removing at least some TILs; and (f) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0179]

[0376] The present invention provides (a) obtaining tumor tissue from a patient, the tumor tissue comprising TILs; (b) incubating the tumor tissue in storage medium at 2°C to 8°C for 30 minutes in storage medium; (c) storing tumor tissue in a frozen state; (d) thawing the tumor tissue; (e) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (f) removing at least a plurality of TILs; and (g) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0180]

[0377] In some embodiments, the method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, the tumor tissue comprising TILs; (b) storing the tumor tissue in a frozen state, the method for storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (iv) refrigerating the container; and (v) storing the container so that it remains frozen; including; (c) thawing the container; (d) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (e) removing at least some TILs; and (f) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas-permeable container to provide an expanded number of TILs. Includes:

[0181]

[0378] In some embodiments, the method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, the tumor tissue comprising TILs; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable container containing a storage medium; (d) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (e) Freezing the container; (f) storing the container so that it remains frozen; (g) thawing the container; (h) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (i) removing at least a plurality of TILs; and (j) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas-permeable container to provide an expanded number of TILs. Includes:

[0182]

[0379] In some embodiments, the tumor tissue is washed with physiologically buffered isotonic saline. In some embodiments, the washing comprises three successive washes of at least 3 minutes each, with the physiologically buffered isotonic saline being replaced after each successive wash. In some embodiments, the physiologically buffered isotonic saline comprises Hank's Balanced Salt Solution (HBSS). In some embodiments, the physiologically buffered isotonic saline comprises Tris-buffered saline (TBS). In some embodiments, the physiologically buffered isotonic saline comprises phosphate-buffered saline (PBS). In some embodiments, the physiologically buffered isotonic saline comprises Dulbecco's Phosphate-buffered Saline (DPBS). In some embodiments, the physiologically buffered isotonic saline in one successive wash may be a different physiologically buffered isotonic saline from that used in one or more other successive washes.

[0183]

[0380] In some embodiments, the thawing step comprises immersing the container in a 37° C. water bath for about 5 minutes.

[0184]

[0381] In some embodiments, the freezing step comprises freezing the container at about −125° C. to about −195° C. In some embodiments, the container is frozen at a temperature of about −125° C. to about −150° C.; in some embodiments, the container is frozen at a temperature of about −125° C. to about −145° C.; in further embodiments, the container is frozen at a temperature of about −135° C.

[0185]

[0382] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney 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.

[0186]

[0383] In some embodiments, the method further comprises splitting between the first expansion culture (or first culture) step and the second expansion culture (or second culture) step. In one embodiment, the splitting occurs on about day 16. In some embodiments, the first expansion culture (or first culture) step is completed in about 11 days. In other embodiments, the first expansion culture (or first culture) step is completed in about 7 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 7 days, and the second expansion culture (or second culture) step is completed in about 14 days. In some embodiments, steps (d) to (f) or steps (h) to (j), as applicable, are completed in about 22 days. In still other embodiments, steps (d) through (f) or steps (h) through (j), as applicable, are completed in about 21 days; in some embodiments, steps (d) through (f) or steps (h) through (j), as applicable, are completed in about 20 days; in some embodiments, steps (d) through (f) or steps (h) through (j), as applicable, are completed in about 16 days.

[0187]

[0384] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) obtaining tumor tissue from a subject, the tumor tissue comprising TILs; (b) storing the tumor tissue in a frozen state, the method for storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 to about 70 minutes; (iv) freezing the container using the vapor phase of liquid nitrogen; and (v) Storing the container in the vapor phase at liquid nitrogen temperature. including; (c) thawing the tumor tissue; (d) adding tumor tissue to a closed system; (e) performing a first expansion culture by culturing a first TIL population from the tumor tissue in a cell culture medium having IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed container providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (d) to step (e) occurs without opening the system; (f) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (e) to step (f) occurs without opening the system; (g) recovering the therapeutic TIL population obtained from step (f), wherein the transition from step (f) to step (g) occurs without releasing the system; (h) transferring the TIL population collected in step (g) to an infusion bag, wherein the transition from step (g) to (h) occurs without opening the system. (i) cryopreserving the infusion bag containing the TIL population collected in step (h) using a cryopreservation process; and (j) administering a therapeutically effective dose of the third population of TILs to the subject from the infusion bag in step (i). Includes:

[0188]

[0385] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) obtaining tumor tissue from a subject, the tumor tissue comprising TILs; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable container containing a storage medium; (d) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 to about 70 minutes; (e) freezing the container using the vapor phase of liquid nitrogen; (f) storing the container in the vapor phase at liquid nitrogen temperatures; (g) thawing the tumor tissue; (h) adding tumor tissue to a closed system; (i) conducting a first expansion culture in a closed system by culturing a first TIL population from the tumor tissue in a cell culture medium having IL-2 to generate a second TIL population, wherein the first expansion culture is conducted in a closed vessel providing a first gas permeable surface area, the first expansion culture is conducted for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (i) to step (j) occurs without opening the system; (k) recovering the therapeutic TIL population obtained from step (j), wherein the transition from step (j) to step (k) occurs without releasing the system; (l) transferring the therapeutic TIL population collected in step (k) to an infusion bag, wherein the transition from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag containing the therapeutic TIL population from step (l) using a cryopreservation process; (o) thawing the therapeutic TIL population in the infusion bag from step (m); and (p) administering a therapeutically effective dose of the therapeutic TIL population to the subject from the infusion bag in step (o). Includes:

[0189]

[0386] In some embodiments, the method for expanding TILs from frozen tumor tissue comprises: (a) storing tumor tissue in a frozen state, the method of storing tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (iv) refrigerating the container; and (v) storing the container so that it remains frozen; including; (b) thawing the container; (c) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (d) removing at least a plurality of TILs; and (e) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas-permeable container to provide an expanded number of TILs. Includes:

[0190]

[0387] In some embodiments, the method for expanding TILs from frozen tumor tissue comprises: (a) Obtaining tumor tissue from the patient; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable container containing a storage medium; (d) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (e) Freezing the container; (f) storing the container so that it remains frozen; (g) thawing the container; (h) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (i) removing at least a plurality of TILs; and (j) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas-permeable container to provide an expanded number of TILs. Includes:

[0191]

[0388] In some embodiments, the fresh tumor tissue is trimmed to a diameter of 1.5 mm to 6 mm and a thickness of about 1.5 mm to about 6 mm, while in other embodiments, the fresh tumor tissue is trimmed to about 6 mm x 6 mm x 6 mm.

[0192]

[0389] 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 at about 0.25 μg / mL to about 2.5 μg / mL. In some embodiments, the antifungal agent is fungin and is present at about 5 μg / mL to about 50 μg / mL.

[0193]

[0390] In some embodiments, the fresh tumor tissue is first washed with Hank's Balanced Salt Solution (HBSS) before performing the trimming step of the method, hi some embodiments, the washing comprises at least three successive washes of at least 3 minutes each, and the HBSS is changed after each wash.

[0194]

[0391] In some embodiments, the thawing step comprises immersing the container in a 37° C. water bath for about 5 minutes.

[0195]

[0392] In some embodiments, the freezing step comprises freezing the container at about −125° C. to about −195° C. In some embodiments, the container is frozen at about −125° C. to about −150° C.; in some embodiments, the container is frozen at about −125° C. to about −145° C.; in further embodiments, the container is frozen at about −135° C.

[0196]

[0393] In some embodiments, the method is performed using a fresh tumor sample from a human subject.

[0197]

[0394] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney 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.

[0198]

[0395] In some embodiments, the method further comprises splitting between the first expansion culture (or first culture) step and the second expansion culture (or second culture) step. In one embodiment, the splitting occurs on day 16. In some embodiments, the first culture step is completed in about 11 days. In other embodiments, the first expansion culture (or first culture) step is completed in about 7 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 7 days, and the second expansion culture (or second culture) step is completed in about 14 days. In some embodiments, steps (c) to (e) or steps (h) to (j), as applicable, are completed in about 22 days. In still other embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are completed in 21 days; in some embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are completed in about 20 days; in some embodiments, steps (c) through (e) or steps (h) through (j), as applicable, are completed in about 16 days.

[0199]

[0396] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) storing tumor tissue in a frozen state, the method of storing tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 to about 70 minutes; (iv) freezing the container using the vapor phase of liquid nitrogen; and (v) Storing the container in the vapor phase at liquid nitrogen temperature. including; (b) thawing the tumor tissue; (c) adding tumor fragments to a closed system; (d) performing a first expansion culture by culturing the first TIL population in a cell culture medium having IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed vessel providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system; (f) recovering the therapeutic TIL population obtained from step (f), wherein the transition from step (e) to step (f) occurs without releasing the system; (g) transferring the TIL population collected in step (f) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) cryopreserving the infusion bag containing the TIL population collected in step (g) using a cryopreservation process; and (i) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the subject. Includes:

[0200]

[0397] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) obtaining tumor tissue from a subject; (b) trimming the tumor tissue to remove excess non-tumor tissue; (c) placing the tumor tissue in a closable container containing a storage medium; (d) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 to about 70 minutes; (e) freezing the container using the vapor phase of liquid nitrogen; (f) storing the container in the vapor phase at liquid nitrogen temperatures; (g) thawing the tumor tissue; (h) adding tumor tissue to a closed system; (i) performing a first expansion culture by culturing a first population of TILs from the tumor tissue in a cell culture medium having IL-2 to generate a second population of TILs, wherein the first expansion culture is performed in a closed container that provides a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second population of TILs, the second population of TILs being at least 50-fold more numerous than the first population of TILs, and the transition from step (h) to step (i) occurs without opening the system; (j) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (i) to step (j) occurs without opening the system; (k) recovering the therapeutic TIL population obtained from step (j), wherein the transition from step (j) to step (k) occurs without releasing the system; (l) transferring the therapeutic TIL population collected in step (k) to an infusion bag, wherein the transition from step (k) to (l) occurs without opening the system; (m) cryopreserving the infusion bag containing the therapeutic TIL population from step (l) using a cryopreservation process; (n) thawing the therapeutic TIL population in the infusion bag from step (m); and (o) administering a therapeutically effective dose of the therapeutic TIL population to the subject from the infusion bag in step (n). Includes:

[0201]

[0398] 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, resistant melanoma, metastatic melanoma, and non-small cell lung cancer. The histology of solid tumors comprises interdependent tissue compartments, including parenchyma (cancer cells), and supporting stromal cells in which cancer cells may disperse and provide a supportive microenvironment.

[0202]

[0399] The present invention provides (a) obtaining tumor tissue from a patient, the tumor tissue comprising TILs; (b) storing the tumor tissue in a frozen state, wherein the method of storing the tumor tissue comprises incubating the tumor tissue in a storage medium at a temperature ranging from about 2°C to about 8°C for about 20 to about 70 minutes prior to freezing; (c) thawing the tumor tissue; (d) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (e) removing at least some TILs; and (f) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0203]

[0400] The present invention provides (a) obtaining tumor tissue from a patient, the tumor tissue comprising TILs; (b) incubating the tumor tissue in storage medium at a temperature ranging from about 2°C to about 8°C for about 20 to 70 minutes; (c) storing tumor tissue in a frozen state; (d) thawing the tumor tissue; (e) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (f) removing at least a plurality of TILs; and (g) expanding the TILs in a second cell culture medium containing cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 in a gas-permeable container to provide an expanded number of TILs. The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, comprising:

[0204]

[0401] In some embodiments, the method for expanding TILs from frozen tumor tissue comprises: (a) obtaining tumor tissue from a patient, the tumor tissue comprising TILs; (b) storing the tumor tissue in a frozen state, the method for storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 30 minutes; (iv) refrigerating the container; and (v) storing the container so that it remains frozen; including; (c) thawing the container; (d) treating the tumor tissue in the gas-permeable container with a first cell culture medium and interleukin-2 (IL-2) to provide TILs; (e) removing at least some TILs; and (f) expanding the TILs in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas-permeable container to provide an expanded number of TILs. Includes:

[0205]

[0402] In some embodiments, the fresh tumor tissue is first washed with Hank's Balanced Salt Solution (HBSS) before performing the tissue trimming step. In some embodiments, the washing comprises at least three consecutive washes of at least 3 minutes each, with the HBSS being replaced after each wash.

[0206]

[0403] In some embodiments, the thawing step comprises immersing the container in a 37° C. water bath for about 5 minutes.

[0207]

[0404] In some embodiments, the freezing step comprises freezing the container at a temperature of about −125° C. to about −195° C. In some embodiments, the container is frozen at a temperature of about −125° C. to about −150° C.; in some embodiments, the container is frozen at a temperature of about −125° C. to about −145° C.; in further embodiments, the container is frozen at a temperature of about −135° C.

[0208]

[0405] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney 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.

[0209]

[0406] In some embodiments, the method further comprises splitting between the first expansion culture (or first culture) step and the second expansion culture (or second culture) step. In one embodiment, the splitting occurs on day 16. In some embodiments, the first culture step is completed in about 11 days. In other embodiments, the first expansion culture (or first culture) step is completed in about 7 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 7 days, and the second expansion culture (or second culture) step is completed in about 14 days. In some embodiments, steps (d) to (f) or steps (e) to (g), as applicable, are completed in about 22 days. In still other embodiments, steps (d) through (f) or steps (e) through (g), as applicable, are completed in about 21 days; in some embodiments, steps (d) through (f) or steps (e) through (g), as applicable, are completed in about 20 days; in some embodiments, steps (d) through (f) or steps (e) through (g), as applicable, are completed in about 16 days.

[0210]

[0407] In some embodiments, the present invention provides a method of treating cancer for a human subject in need thereof, such method comprising: (a) obtaining tumor tissue from a subject, the tumor tissue comprising TILs; (b) storing the tumor tissue in a frozen state, the method for storing the tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) placing the tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and storage medium at a temperature ranging from about 2°C to about 8°C for about 20 minutes to about 70 minutes; (iv) freezing the container using the vapor phase of liquid nitrogen; and (v) Storing the container in the vapor phase at liquid nitrogen temperature. including; (c) thawing the tumor tissue; (d) adding tumor tissue to a closed system; (e) performing a first expansion culture by culturing a first TIL population from the tumor tissue in a cell culture medium having IL-2 to generate a second TIL population, wherein the first expansion culture is performed in a closed container providing a first gas permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain a second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and the transition from step (d) to step (e) occurs without opening the system; (f) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the second expansion culture is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion culture is performed in a closed vessel providing a second gas-permeable surface area, and the transition from step (e) to step (f) occurs without opening the system; (g) recovering the therapeutic TIL population obtained from step (f), wherein the transition from step (f) to step (g) occurs without releasing the system; (h) transferring the TIL population collected in step (g) to an infusion bag, wherein the transition from step (g) to (h) occurs without opening the system. (i) cryopreserving the infusion bag containing the TIL population collected in step (h) using a cryopreservation process; and (j) administering a therapeutically effective dose of the third population of TILs to the subject from the infusion bag in step (i). Includes:

[0211]

[0408] In some embodiments, the fresh tumor tissue is first washed with Hank's Balanced Salt Solution (HBSS) prior to performing step (i) of the tumor tissue storage method, in some embodiments, the washing comprises at least three successive washes of at least 3 minutes each, and the HBSS is replaced after each wash.

[0212]

[0409] In some embodiments, step (c) comprises immersing the container in a 37° C. water bath for about 5 minutes.

[0213]

[0410] In some embodiments, step (iv) of the tumor tissue storage method comprises freezing the container at a temperature of about −125° C. to about −195° C. In some embodiments, the container is frozen at a temperature of about −125° C. to about −150° C.; in some embodiments, the container is frozen at a temperature of about −125° C. to about −145° C.; in further embodiments, the container is frozen at a temperature of about −135° C.

[0214]

[0411] In some embodiments, the tumor tissue is selected from the group consisting of melanoma tumor tissue, head and neck tumor tissue, breast tumor tissue, kidney 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.

[0215]

[0412] In some embodiments, the method further comprises splitting between the first expansion culture (or first culture) step and the second expansion culture (or second culture) step. In one embodiment, the splitting occurs on day 16. In some embodiments, the first culture step is completed in about 11 days. In other embodiments, the first expansion culture (or first culture) step is completed in about 7 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 7 days and the second expansion culture (or second culture) step is completed in about 14 days. In some embodiments, steps (e) to (g) are completed in about 22 days. In still other embodiments, steps (e) to (g) are completed in about 21 days; in some embodiments, steps (e) to (g) are completed in about 20 days; and in some embodiments, steps (e) to (g) are completed in about 16 days.

[0216]

[0413] In some embodiments, the first expansion culture (or first culture) step is completed in about 5 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 3 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 5 days, and the second expansion culture (or second culture) step is completed in about 11 days. In yet other embodiments, the first expansion culture (or first culture) step is completed in about 3 days, and the second expansion culture (or second culture) step is completed in about 13 days. Optionally, the second expansion culture (or second culture) can be split into two or more cultures on or around day 5 or 6 of the second expansion culture (or second culture).

[0217]

[0414] In some embodiments of the above methods for producing or expanding TILs, OKT-3 is present in the medium from day 0.

[0218]

[0415] In some embodiments, the present invention provides any of the aforementioned methods for treating cancer in a subject, optionally modified in the first expansion culture (or first culture) step so that the first expansion culture (or first culture) is completed in about 5 days.

[0219]

[0416] In some embodiments, the present invention provides any of the aforementioned methods for treating cancer in a subject, optionally modified in the first expansion culture (or first culture) step so that the first expansion culture (or first culture) is completed in about 3 days.

[0220]

[0417] In some embodiments, the present invention provides any of the aforementioned methods for treating cancer in a subject, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 5 days, and the second expansion culture (or second culture) is completed in about 11 days.

[0221]

[0418] In some embodiments, the present invention provides any of the aforementioned methods of treating cancer in a subject, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 5 days, the second expansion culture (or second culture) is completed in about 11 days, and the second expansion culture (or second culture) is split into two or more cultures on or about day 5 of the second expansion culture (or second culture).

[0222]

[0419] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, and the second expansion culture (or second culture) is completed in about 13 days.

[0223]

[0420] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, the second expansion culture (or second culture) is completed in about 13 days, and the second expansion culture (or second culture) is split into two or more cultures on or about day 6 of the second expansion culture (or second culture).

[0224]

[0421] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 5 days.

[0225]

[0422] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days.

[0226]

[0423] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 5 days and the second expansion culture (or second culture) is completed in about 11 days.

[0227]

[0424] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 5 days, the second expansion culture (or second culture) is completed in about 11 days, and the second expansion culture (or second culture) is split into two or more cultures on or about day 5 of the second expansion culture (or second culture).

[0228]

[0425] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, and the second expansion culture (or second culture) is completed in about 13 days.

[0229]

[0426] In some embodiments, the present invention provides any of the aforementioned methods for expanding tumor-infiltrating lymphocytes (TILs) from frozen tumor tissue, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, the second expansion culture (or second culture) is completed in about 13 days, and the second expansion culture (or second culture) is split into two or more cultures on or about day 6 of the second expansion culture (or second culture).

[0230]

[0427] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, and the second expansion culture (or second culture) is completed in about 13 days.

[0231]

[0428] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, the second expansion culture (or second culture) is completed in about 13 days, and the second expansion culture (or second culture) is split into two or more cultures on or about day 6 of the second expansion culture (or second culture).

[0232]

[0429] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified in the first expansion culture (or first culture) step so that the first expansion culture (or first culture) is completed in about 5 days.

[0233]

[0430] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified in the first expansion culture (or first culture) step so that the first expansion culture (or first culture) is completed in about 3 days.

[0234]

[0431] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 5 days and the second expansion culture (or second culture) is completed in about 11 days.

[0235]

[0432] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 5 days, the second expansion culture (or second culture) is completed in about 11 days, and the second expansion culture (or second culture) is split into two or more cultures on or about day 5 of the second expansion culture (or second culture).

[0236]

[0433] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, and the second expansion culture (or second culture) is completed in about 13 days.

[0237]

[0434] In some embodiments, the present invention provides any of the aforementioned methods for preparing tumor-infiltrating lymphocytes (TILs) for adoptive T cell therapy, optionally modified so that in the first expansion culture (or first culture) step, the first expansion culture (or first culture) is completed in about 3 days, the second expansion culture (or second culture) is completed in about 13 days, and the second expansion culture (or second culture) is split into two or more cultures on or about day 6 of the second expansion culture (or second culture).

[0238]

[0435] In some embodiments, the present invention provides any of the aforementioned methods, suitably modified so that the first expansion culture (or first culture) is performed in a defined medium.

[0239]

[0436] In some embodiments, the present invention provides any of the aforementioned methods, suitably modified so that the second expansion culture (or second culture) is performed in a defined medium.

[0240]

[0437] In some embodiments, the present invention provides any of the aforementioned methods, suitably modified so that the first expansion culture (or first culture) and the second expansion culture (or second culture) are carried out in the same or different defined media.

[0241] How TILs are manufactured

[0438] There are various methods for expanding TILs known to those 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," the disclosure of which is incorporated herein by reference, teaches a simplified method for producing TILs for clinical use. Jin et al. teach a first TIL culture followed by a rapid expansion (REP) protocol, which, in combination, allows one skilled in the art to produce clinically useful quantities of TILs. In some embodiments, the present invention provides a method for producing TILs, comprising the steps of cryopreserving a tumor, thawing the tumor, and carrying out the process described in Jin et al. Briefly, this process includes the following steps: TILs are first prepared by cryopreserving tumor fragments (approximately 1-8 mm) obtained by enzymatic tumor digestion and sharp dissection. 3Tumors can be cultured from 1000-well cultures. Tumor digests are produced by incubation in enzyme medium (RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). Immediately after placing the tumor in the enzyme medium, it is mechanically dissociated for approximately 1 minute. The material is then incubated for 30 minutes at 37°C in 5% CO2, 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. After the third mechanical disruption, if large tissue fragments are present, the sample may be subjected to one or two additional mechanical dissociations, with or without an additional 30-minute incubation at 37°C in 5% CO2. At the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells. If TIL cultures are initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, NY), each well contains 1 × 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 1 tumor digestive cell or approximately 1-8 mm 3 The CM contains RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and approximately 10 μg / mL gentamicin. The volume is 40 mL and the CM is 10 cm. 2 When cultures were initiated in gas-permeable flasks (G-Rex 10; Wilson Wolf Manufacturing, New Brighton, MN) with gas-permeable silicone bottoms, each flask contained 10–40 × 10 cells in 10–40 mL of CM containing IL-2. 6Viable tumor digested cells or 5-30 tumor fragments are loaded into the G-Rex 10-well and 24-well plates. Both the plates are incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium is removed and replenished with fresh CM and IL-2. From day 5 onwards, half of the medium is replaced every 2-3 days. TIL REP is performed using a T-175 flask with a gas-permeable bag or a gas-permeable G-Rex flask. For TIL REP in a T-175 flask, 1 x 10 cells suspended in 150 mL of medium are used. 6 TILs were added to each T-175 flask. The TILs were cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells were cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks were incubated at 37°C under 5% CO2. Half of the medium was replaced on day 5 with 50 / 50 medium containing 3000 IU / mL IL-2. On day 7, cells from two T-175 flasks were combined in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 were added to the 300 mL TIL suspension. The number of cells in each bag was counted daily or every other day, and fresh medium was added to keep the cell count at 0.5–2.0 × 10 6 cells / mL. 100cm 2 For TIL REP in a 500 mL volumetric flask with gas-permeable silicone bottom (G-Rex 100, Wilson Wolf), 5 × 10 cells were cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. 6 ~10×10 6TILs were cultured at a 1:100 ratio with irradiated allogeneic PBMCs. G-Rex 100 flasks were incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant was removed and placed in a centrifuge bottle. The flask was centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet was resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL IL-2 and added back to the original G-Rex 100 flask. If TILs were to be continuously expanded in the G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 flask were suspended in the 300 mL of medium present in each flask. The cell suspension was divided into three 100 mL aliquots, which were then used to seed three G-Rex 100 flasks. 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to each flask. The G-Rex 100 flasks were incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 was added to each G-Rex 100 flask. Cells were harvested on day 14 of culture.

[0242]

[0439] In some embodiments, the expansion method according to the present invention involves preparing expansion cultures of cells from frozen tumors. Tumor samples are collected from patients and cryopreserved according to the methods disclosed herein. When the cells are ready for expansion, for a process performed at 1 / 100 scale, approximately six frozen tumor fragments, 2-3 mm in diameter, are thawed for 5 + 1 minutes using a 37°C water bath and washed with sterile Hank's balanced salt solution (HBSS) supplemented with gentamicin. The fragments are then placed in G-Rex 10M flasks (Wilson Wolf Mfg., New Brighton, MN) or other gas-permeable containers with CM1 medium or serum-free or defined medium containing 6,000 IU / mL rhIL-2. These pre-REP (or first expansion) cultures are incubated in a 37°C incubator for 5 days. On day 5, pre-REP cells were harvested, and the REP (or second expansion) was performed by culturing 10% of the pre-REP TILs at 25 × 10 in CM2 medium or serum-free or defined medium containing 3,000 IU / mL rhIL-2 and 30 ng / mL OKT-3 in a G-Rex 5M flask (Wilson Wolf Mfg., New Brighton, MN) or other gas-permeable container. 6 or 50 x 10 6 On day 10, the culture is expanded into a G-Rex 5M flask or 10 x 10 PBMC feeder cells. 6 The cells are split into another gas-permeable container containing no more than 100 cells. The split culture is incubated for an additional 6 days in CM4 medium or other serum-free or defined medium containing 3,000 IU / mL rhIL-2. On day 16, the cells are harvested and frozen using CryoStor 10 (Biolife, USA). This process is referred to herein as the "Early REP Method 1" process or "ER-1."

[0243]

[0440] In some embodiments, the expansion method according to the present invention involves preparing expansion cultures of cells from frozen tumors. Tumor samples are collected from patients and cryopreserved according to the methods disclosed herein. When the cells are ready for expansion, for a process performed at 1 / 100 scale, approximately six frozen tumor fragments, 2-3 mm in diameter, are thawed for 5 + 1 minutes using a 37°C water bath and washed with sterile Hank's balanced salt solution (HBSS) supplemented with gentamicin. The fragments are then placed in G-Rex 10M flasks (Wilson Wolf Mfg., New Brighton, MN) or other gas-permeable containers with CM1 medium or approximately serum-free or defined medium containing 6,000 IU / mL rhIL-2. These pre-REP (or first expansion) cultures are incubated in a 37°C incubator for three days. On day 3, pre-REP cells were harvested, and the REP (or second expansion) was performed by culturing 10% of the pre-REP TILs at 25 × 10 in CM2 medium or serum-free or defined medium containing 3,000 IU / mL rhIL-2 and 30 ng / mL OKT-3 in a G-Rex 5M flask (Wilson Wolf Mfg., New Brighton, MN) or other gas-permeable container. 6 or 50 x 10 6 On day 9, the culture is expanded to a G-Rex 5M flask or 10 x 10 PBMCs by co-culturing with 10 x 10 feeder cells. 6 The cells are split into another gas-permeable container containing no more than 100 cells. The split culture is incubated for an additional 7 days in CM4 medium or other serum-free or defined medium 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."

[0244]

[0441] In some embodiments, the present invention provides a method for producing TILs, comprising the steps of cryopreserving tumors, thawing the tumors, and performing the following process: TILs can be produced by rapid expansion using in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with a cancer antigen (including one or more antigenic portions thereof, such as epitopes or cells), 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 (preferably IL-2). In vitro-induced TILs can also be rapidly expanded by restimulation with the same cancer antigen or antigens pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be rapidly expanded by, for example, irradiated autologous lymphocytes or by irradiating HLA-A2-binding peptides. +TILs can be restimulated with allogeneic lymphocytes and IL-2. TILs can be selected for highly active recognition of any of the unique antigens generated as a result of the estimated 10,000 genetic mutations encoded by each tumor cell genome. However, the antigen need not be unique. T cells can be selected for highly active recognition of one or more antigens of the cancer, including antigenic portions of one or more antigens, such as epitopes, or cells of the cancer. "Cancer antigen" and "cancer antigen" are intended to encompass all of the aforementioned antigens. When the cancer is melanoma, such as metastatic melanoma, TILs are preferably selected for highly active recognition of "unique" or patient-specific antigens derived from tumor-encoded mutations, such as MART-1 (e.g., MART-1:26-35(27L)), gp100 (e.g., gp100:209-217(210M)), or tumor-encoded mutations. Other suitable melanoma antigens that can be selected for highly active recognition by TILs include, but are not limited to, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, and MAGE. For the treatment of lung cancer, breast cancer, colon cancer, prostate cancer, etc., antigens such as NY-ESO-1, telomerase, p53, HER2 / neu, carcinoembryonic antigen, or prostate-specific antigen can be used to select for highly active recognition by TILs. TILs that can be selected include, but are not limited to, tyrosinase and tyrosinase-related proteins.

[0245]

[0442] IL-2-based TIL expansion followed by the "rapid expansion process" (REP) is becoming the preferred method for TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-fold expansion of TILs in a 14-day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs) as feeder cells, often from multiple donors, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. Cryopreserved tumor fragments are a suitable starting point for initial or primary cultures to produce TILs for therapeutic or other purposes.

[0246]

[0443] An example of a TIL process known as Process 2A that includes some of these features is shown 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 in Figure 1.

[0247]

[0444] As discussed herein, the invention may include steps relating to restimulating cryopreserved TILs prior to transplantation into a patient to increase their metabolic activity, and therefore their relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are typically obtained from a patient sample and manipulated to expand their numbers prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0248]

[0445] In some embodiments, TILs can be cryopreserved and thawed for administration to a patient. After thawing, the TILs can be restimulated to enhance their metabolism before infusion into a patient.

[0249]

[0446] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion culture (including a process referred to as Pre-REP and shown as step A in FIG. 1 ) is shortened to 3-14 days, and the second expansion culture (including a process referred to as REP and shown as step B in FIG. 1 ) is shortened to 7-14 days. In some embodiments, the first expansion culture (e.g., the expansion culture described as step B in FIG. 1 ) is shortened to 11 days, and the second expansion culture (e.g., the expansion culture described as step D in FIG. 1 ) is shortened to 11 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the combined first and second expansion cultures (e.g., the expansion cultures described as steps B and D in FIG. 1 ) are shortened to 22 days.

[0250]

[0447] The "step" designations A, B, C, etc. below refer to Figure 1 and to specific embodiments described herein. The order of steps below and in Figure 1 is exemplary, and any combination or order of steps, as well as additional steps, repeated steps, and / or omission of steps, is contemplated by the present application and methods disclosed herein.

[0251] Step A. Obtain patient tumor samples

[0448] Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally have phenotypic and metabolic parameters determined as indicators of TIL health.

[0252]

[0449] Patient tumor samples may be obtained using methods known in the art, generally by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples may be from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples may also be from liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors may be of any cancer type, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, renal cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these tumors have been reported to have particularly high levels of TILs.

[0253]

[0450] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells), and supporting stromal cells in which cancer cells may disperse and provide a supportive microenvironment.

[0254]

[0451] The term "hematologic malignancies" refers to mammalian cancers and tumors of the hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematologic malignancies are also referred to as "liquid tumors." Hematologic malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B cells.

[0255]

[0452] Once obtained, tumor samples are typically cut into 1 to approximately 8 mm sections using sharp dissection. 3 fragmented into small pieces of about 2-3 mm 3 are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociation agent). Tumor digests can be produced by placing the tumor in the enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art may also be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for the methods of expanding TILs or treating cancer.

[0256]

[0453] The harvested cell suspension is generally referred to as a "primary cell population" or a "freshly harvested" cell population.

[0257]

[0454] In some embodiments, fragmentation comprises physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In one embodiment, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0258]

[0455] In some embodiments, if the tumor is a solid tumor, for example, after obtaining a tumor sample in step A (provided in FIG. 1), the tumor is subjected to physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm. 3 In some embodiments, the plurality of pieces has a volume of about 1300 mm 3 ~approx. 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces having a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 g to about 1.5 g. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0259]

[0456] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm. In embodiments where the tumor tissue is first frozen before initiation of culture, the tumor is approximately 6 mm x 6 mm x 6 mm.

[0260]

[0457] In some embodiments, the tumor is resected to minimize the amount of bleeding, necrosis, and / or fatty tissue on each segment. In some embodiments, the tumor is resected to minimize the amount of bleeding tissue on each segment. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each segment. In some embodiments, the tumor is resected to minimize the amount of fatty tissue on each segment.

[0261]

[0458] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are created by incubation in an enzyme medium, such as, 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 the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C under 5% CO2 for 30 minutes, after which it may be mechanically disrupted again for approximately 1 minute. After another 30-minute incubation at 37°C under 5% CO2, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue debris was present after the third mechanical disruption, the sample was subjected to one or two additional rounds of mechanical separation, with or without an additional 30-minute incubation at 37° C. under 5% CO. In some embodiments, if the cell suspension at the end of the final incubation contained a large number of red blood cells or dead cells, density gradient separation using Ficoll may be performed to remove such cells.

[0262]

[0459] In some embodiments, the harvested cell suspension before the first expansion step is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0263]

[0460] In some embodiments, the cells may optionally be frozen after sample collection and stored frozen before proceeding to expansion as described in step B, described in more detail below and illustrated in FIG. 1.

[0264] Step B: First expansion culture

[0461] In some embodiments, the methods provide for obtaining young TILs that are capable of increasing replication cycles upon administration to a subject / patient, and may therefore provide an additional therapeutic advantage over mature TILs (e.g., "mature TILs" that have undergone more rounds of ex vivo replication before administration to a subject / patient). Characteristics of young TILs have been described in the literature. For example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010), Huang et al., J Immunother, 28(3):258-267 (2005), Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013), Besser et al., J Immunother 32:415-423 (2009), Robbins, et al., J Immunol 2004; 173:7125-7130, Shen et al., J Immunother, 30:123-129 (2007), Zhou, et al., J Immunother, 28:53-62 (2005), and Tran, et al., J Immunother, 31:742-751 (2008), all of which are incorporated herein by reference in their entireties.

[0265]

[0462] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but numerous gene segments. The V (variable), D (diversity), J (joining), and C (constant) gene segments determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly collected TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, as illustrated in FIG. 5 and / or FIG. 6, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly collected TILs and / or TILs prepared using a method referred to as Process 1C. In some embodiments, TILs obtained in the first expansion culture exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCR α / β) is increased.

[0266]

[0463] After dissection or digestion of tumor fragments, for example, 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, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 Bulk TIL cells are obtained.

[0267]

[0464] In a preferred embodiment, as described below and herein, expansion of TILs can be performed using an initial bulk TIL expansion step (e.g., such as that described in step B of FIG. 1, which may include a process referred to as pre-REP), followed by a second expansion (step D, which includes a process referred to as a rapid expansion protocol (REP) step), as described below under step D and herein, followed by optional cryopreservation, and then a second step D (which includes a process referred to as a restimulation REP step). TILs obtained by this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0268]

[0465] In an embodiment, when TIL cultures are initiated in 24-well plates, e.g., using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), each well contains 1 x 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 Tumor digest cells or 1 tumor fragment can be seeded. In some embodiments, the tumor fragment is about 1 mm 3 ~10mm 3 is.

[0269]

[0466] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 μg / mL gentamicin. The culture is grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10 x 10 cells in 10-40 mL of IL-2-containing CM. 6 ~40×10 6 Live tumor digest cells or 5–30 tumor fragments were loaded into G-Rex10 and 24-well plates. Both were incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onward, half of the medium was replaced every 2–3 days.

[0467]

[0270]

[0468] After preparation of 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, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2 (or in some cases in the presence of an aAPC cell population, as outlined herein). This primary cell population is cultured for several days, generally 10-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion culture contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg of IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example E. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL IL-2 to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL IL-2 to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL of IL-2, between about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL, or about 8000 IU / mL of IL-2.

[0271]

[0469] In some embodiments, the first expansion culture medium comprises about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium comprises about 500 IU / mL IL-15 to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium comprises about 400 IU / mL IL-15 to about 100 IU / mL IL-15. In some embodiments, the first expansion culture medium comprises about 300 IU / mL of IL-15, to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In one preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0272]

[0470] In some embodiments, the first expansion culture medium 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 medium comprises about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium comprises about 12 IU / mL of IL-21, between about 0.5 IU / mL. In some embodiments, the first expansion culture medium comprises about 10 IU / mL of IL-21, between about 0.5 IU / mL. In some embodiments, the first expansion culture medium comprises about 5 IU / mL of IL-21, between about 1 IU / mL. In some embodiments, the first expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[0273]

[0471] In one embodiment, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of the OKT-3 antibody. In one embodiment, the cell culture medium comprises an OKT-3 antibody at a concentration of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain an OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0274]

[0472] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL in the cell culture medium.

[0275]

[0473] In some embodiments, in addition to one or more TNFRSFs, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and an OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0276]

[0474] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The culture is grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10 x 10 cells in 10-40 mL of IL-2-containing CM. 6 ~40×10 6 Live tumor digest cells or 5-30 tumor fragments were loaded. Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onwards, half of the medium was replaced every 2-3 days. In some embodiments, the CM is CM1 as described in the Examples (see Example 1). In some embodiments, the first expansion culture is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

[0277]

[0475] In some embodiments, as discussed in the Examples and Figures, the first expansion culture (e.g., including the process described in step B of Figure 1, which may include what may be referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples, including, for example, Figures 4 and 5 and the Examples, including, for example, the Example described in step B of Figure 1, the first expansion culture (e.g., including the process described in step B of Figure 1, which may be referred to as pre-REP) is shortened to 7-14 days. In some embodiments, the first expansion culture in step B is shortened to 10-14 days. In some embodiments, as discussed in the Examples, for example, the expansion culture described in step B of Figure 1, the first expansion culture is shortened to 11 days.

[0278]

[0476] In some embodiments, the first TIL expansion culture may be continued 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 culture may be continued for 1 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 2 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 3 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 8 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 11 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 12 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 13 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 14 days. In some embodiments, the first TIL expansion culture may be continued for 1 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 2 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 3 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 8 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 11 days.In some embodiments, the first TIL expansion culture may continue for 11 days.

[0279]

[0477] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination in the first expansion culture. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and optional combinations thereof, may be included in the first expansion culture, including in step B process, e.g., according to FIG. 1 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination in the first expansion culture. In some embodiments, IL-2, IL-15, and IL-21, and optional combinations thereof, may be included in step B process, e.g., according to FIG. 1 and as described herein.

[0280]

[0478] In some embodiments, as discussed in the Examples and Figures, the first expansion culture (including, for example, the process described in step B of FIG. 1, referred to as Pre-REP) process is shortened to 3-14 days. In some embodiments, the first expansion culture in step B is shortened to 7-14 days. In some embodiments, the first expansion culture in step B is shortened to 10-14 days. In some embodiments, the first expansion culture is shortened to 11 days.

[0281]

[0479] In some embodiments, the first expansion, e.g., step B according to FIG. 1, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion, as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0282] Step C: Transition from the first expansion culture to the second expansion culture

[0480] In some cases, the bulk TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B as shown in Figure 1, may be immediately cryopreserved using the protocols discussed herein below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, may be subjected to a second expansion (which may include an expansion sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, when genetically modified TILs are used therapeutically, the first TIL population (which may also be referred to as the bulk TIL population) or the second TIL population (which may, in some embodiments, include a population referred to as the REP TIL population) may be subjected to genetic modification for an appropriate treatment before expansion or after the first expansion but before the second expansion.

[0283]

[0481] In some embodiments, TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 1 ) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 1 ) are not stored but proceed directly to the second expansion culture. In some embodiments, TILs obtained from the first expansion culture are not cryopreserved after the first expansion culture and before the second expansion culture. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 3 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 10 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 14 days after fragmentation.

[0284]

[0482] In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 1 day to 14 days after fragmentation. In some embodiments, the first TIL expansion culture may continue for 2 days to 14 days. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 3 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 4 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 5 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 6 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 8 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 9 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 10 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 11 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 12 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 13 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 1 to 11 days after fragmentation.In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 2 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 3 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 4 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 5 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 6 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 7 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 8 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 9 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 10 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 11 days after fragmentation.

[0285]

[0483] In some embodiments, the TILs are not stored after the first expansion and before the second expansion, and the TILs proceed directly to the second expansion (e.g., in some embodiments, no storage occurs during the transition from step B to step D, as shown in FIG. 1). In some embodiments, the transfer occurs in a closed system, as described herein. In some embodiments, the TILs from the first expansion are TILs from a second TIL population and proceed directly to the second expansion without a transfer period.

[0286]

[0484] In some embodiments, the transition from the first expansion culture to the second expansion culture, e.g., step C according to FIG. 1, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0287] cytokines

[0485] The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as is known in the art.

[0288]

[0486] Alternatively, it is further possible to use combinations of cytokines in the rapid and / or secondary expansion of TILs, such as combinations of two or more of IL-2, IL-15, and IL-21, generally as outlined in WO 2015 / 189356 and WO 2015 / 189357, which are expressly incorporated by reference in their entireties. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15, IL-21 and IL-2, and IL-15 and IL-21, the latter finding particular use in many embodiments. As described therein, the use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells.

[0289] Step D: Second expansion culture

[0487] In some embodiments, the TIL cell population is expanded in number after harvesting and initial bulk processing, e.g., following steps A and B, and a transition referred to as step C, as described in FIG. 1. This further expansion is referred to herein as a second expansion, which may include an expansion process commonly referred to in the art as rapid expansion (REP, and the process depicted in FIG. 1, step D). The second expansion can generally be accomplished using culture medium in a gas-permeable vessel containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody.

[0290]

[0488] In some embodiments, the second expansion of TILs or the second TIL expansion culture (which may also be referred to as REP; and may include the process shown in step D of FIG. 1 ) can be performed using any TIL flask or vessel known to those of skill in the art. In some embodiments, the second TIL expansion culture may continue for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion culture may continue for about 7 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 8 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 9 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 10 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 11 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 12 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 13 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 14 days.

[0291]

[0489] In one embodiment, the second expansion can be performed in a gas-permeable container using the methods of the present disclosure (e.g., including the expansion referred to as REP, as well as the process shown in step D of FIG. 1). For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation can include, for example, about 30 ng / ml of an anti-CD3 antibody such as OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA), or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further stimulation in vitro by introducing one or more antigens of the cancer, including antigenic portions thereof, such as one or more epitopes, optionally expressed from a vector, such as human leukocyte antigen A2 (HLA-A2)-binding peptides, e.g., 0.3 μM MART-1:26-35(27L) or gpl 00:209-217(210M), during the second expansion, optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same one or more antigens of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second expansion culture. In some embodiments, the second expansion culture occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0292]

[0490] In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium contains 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL of IL-2.

[0293]

[0491] In one embodiment, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of the OKT-3 antibody. In one embodiment, the cell culture medium comprises an OKT-3 antibody at a concentration of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium does not contain an OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0294]

[0492] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL in the cell culture medium.

[0295]

[0493] In some embodiments, in addition to one or more TNFRSFs, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and an OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0296]

[0494] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination in the second expansion culture. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and optional combinations thereof, may be included in the second expansion culture, including during step D of the process, e.g., according to FIG. 1 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination in the second expansion culture. In some embodiments, IL-2, IL-15, and IL-21, and optional combinations thereof, may be included in the step D of the process, e.g., according to FIG. 1 and as described herein.

[0297]

[0495] In some embodiments, the second expansion culture may be performed 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 culture is performed 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 culture occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0298]

[0496] In some embodiments, the second expansion culture medium comprises about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium comprises about 500 IU / mL IL-15 to about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium comprises about 400 IU / mL IL-15 to about 100 IU / mL IL-15. In some embodiments, the second expansion culture medium comprises about 300 IU / mL of IL-15, to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In one preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0299]

[0497] In some embodiments, the second expansion culture medium 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 medium comprises about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 12 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 10 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 5 IU / mL of IL-21, or about 1 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[0300]

[0498] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion culture and / or second expansion culture is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:50 to 1:300. In one embodiment, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:100 to 1:200.

[0301]

[0499] In one embodiment, REP and / or secondary expansion is performed in flasks by mixing bulk TILs with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. Media replenishment is performed (typically two-thirds via respiration with fresh medium) until the cells are transferred to a separate growth chamber. Separate growth chambers include G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0302]

[0500] In some embodiments, the second expansion culture (which may include a process called the REP process) is shortened to 7-14 days, as discussed in the Examples and Figures. In some embodiments, the second expansion culture is shortened to 11 days.

[0303]

[0501] In one embodiment, the REP and / or second expansion culture may be performed using T-175 flasks and gas permeable bags or gas permeable cultureware (G-Rex flasks) as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42). In some embodiments, the second expansion culture (including expansion cultures referred to as rapid expansion cultures) is performed in a T-175 flask and contains approximately 1 x 10 cells suspended in 150 mL of medium. 6 TILs may be added to each T-175 flask. TILs may be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. T-175 flasks may be incubated at 37°C under 5% CO2. Half of the medium may be replaced on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks may be combined into a 3 L bag, and 300 mL of the TIL suspension may be added with 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2. The number of cells in each bag is counted daily or every two days, and fresh medium is added to increase the cell count to 0.5–2.0 × 10. 6 cells / mL.

[0304]

[0502] In one embodiment, the second expansion culture (which may include the expansion culture referred to as REP; and referenced in step D of FIG. 1 ) may be performed in a 100 cm gas-permeable silicone-bottomed, 500 mL capacity gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) and cultured at 5×10 cells in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL IL-2, and 30 ng / mL anti-CD3 (OKT3). 6 or 10 x 10 6TILs can be cultured with PBMCs. The G-Rex 100 flasks can be incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU / mL IL-2 and added back to the original G-Rex 100 flask. When serially expanding TILs in G-Rex 100 flasks, on day 7, the TILs from each G-Rex 100 can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots that can be used to seed three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to each flask. The G-Rex 100 flasks may be incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 may be added to each G-Rex 100 flask. Cells may be harvested on day 14 of culture.

[0305]

[0503] In one embodiment, the second expansion culture (including the expansion culture referred to as REP) is performed in a flask by mixing bulk TILs with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml medium. In some embodiments, medium changes are performed until the cells are transferred to another growth chamber. In some embodiments, two-thirds of the medium is replaced by respiration with fresh medium. In some embodiments, the other growth chamber includes G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0306]

[0504] In one embodiment, a second expansion culture (including an expansion culture called REP) is performed, which further includes selecting TILs for superior tumor responsiveness. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1, the disclosure of which is incorporated herein by reference, can be used to select TILs for superior tumor responsiveness.

[0307]

[0505] Optionally, after the second expansion (including the expansion referred to as the REP expansion), a cell viability assay can be performed using standard assays known in the art. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows assessment of viability. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

[0308]

[0506] In some embodiments, the second expansion of TILs (including expansions referred to as REPs) 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 contains approximately 1 x 10 6TILs are suspended in approximately 150 mL of medium and added to each T-175 flask. The TILs are cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells are cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks are incubated at 37°C under 5% CO. In some embodiments, the medium is changed on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks are combined into a 3 L bag, and 300 mL of the TIL suspension is added to 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2. The number of cells in each bag may be counted daily or every two days, and fresh medium may be added to increase the cell count to about 0.5 to about 2.0 x 10 6 cells / mL.

[0309]

[0507] In some embodiments, the second expansion culture (including the expansion culture referred to as REP) is 100 cm 2 The cells were cultured in a 500 mL volumetric flask (G-Rex 100, Wilson Wolf) with a gas-permeable silicone bottom (Figure 1) and approximately 5 × 10 cells were cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. 6 or 10 x 10 6TILs are cultured with irradiated allogeneic PBMCs at a 1:100 ratio. G-Rex100 flasks are incubated at 37°C under 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL of IL-2 and added back to the original G-Rex100 flask. In embodiments where TILs are serially expanded in G-Rex100 flasks, on day 7, the TILs in each G-Rex100 are suspended in the 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots, which are used to seed three G-Rex100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to each flask. The G-Rex 100 flasks were incubated at 37°C under 5% CO2. After 4 days, 150 mL of AIM-V containing 3000 IU / mL IL-2 was added to each G-Rex 100 flask. Cells were harvested on day 14 of culture.

[0310]

[0508] Diverse antigen receptors for T and B lymphocytes are produced by somatic recombination of a limited but numerous 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 methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by this met...

Claims

1. 1. A method for cryopreserving tumor tissue for the production of tumor infiltrating lymphocytes (TILs), comprising: (i) fragmenting the tumor tissue; (ii) incubating the fragments in a cryopreservation medium; and (iii) freezing the fragment, the freezing being a flash freezing using the vapor phase of liquid nitrogen. A method comprising:

2. The method of claim 1, wherein the tumor tissue is fragmented into approximately spherical fragments having a diameter between 1.5 mm and 6 mm.

3. The method of claim 2 , wherein the generally spherical pieces have a diameter of 6 mm.

4. The method of claim 2 , wherein the generally spherical pieces have a diameter of 3 mm.

5. 10. The method of claim 1, wherein the tumor tissue is fragmented into approximately rectangular pieces having a shortest dimension of at least 1.5 mm and a longest dimension of at least 6 mm.

6. The method of claim 1, wherein the tumor tissue is fragmented into roughly cubic pieces having side lengths between 1.5 mm and 6 mm.

7. The method of claim 6 , wherein the generally cubic pieces have a side length of 6 mm.

8. The method of claim 6 , wherein the generally cubic pieces have a side length of 3 mm.

9. The method of claim 1 , wherein the tumor tissue is from a dissected tumor.

10. 10. The method of claim 1, wherein the dissected tumor tissue is less than 8 hours old.

11. 2. The method of claim 1, wherein the cryopreservation medium comprises 2% v / v to 15% v / v dimethylsulfoxide (DMSO).

12. 12. The method of claim 11, wherein the cryopreservation medium comprises 10% v / v DMSO.

13. The method of claim 1 , wherein the cryopreservation medium comprises at least one antimicrobial agent.

14. 14. The method of claim 13, wherein the at least one antibacterial agent is gentamicin at a concentration of at least 50 μg / mL.

15. The method of claim 1, wherein the tumor fragments are incubated in the cryopreservation medium for 30 to 80 minutes.

16. The method of claim 1, wherein the tumor fragments are incubated in the cryopreservation medium at a temperature between 2°C and 8°C.

17. The method of claim 1, wherein the tumor tissue is washed with physiological buffered isotonic saline prior to incubation.

18. 18. The method of claim 17, wherein the washing comprises three successive washes of at least 3 minutes each, with the physiological buffered isotonic saline being replaced after each successive wash.

19. 2. The method of claim 1, wherein the freezing is carried out at a temperature in the range of -125°C to -196°C.

20. 20. The method of claim 19, wherein the freezing is carried out at a temperature of from -140°C to -175°C.

21. 21. The method of claim 20, wherein the freezing is performed at a temperature of -145°C.

22. 10. The method of claim 1, further comprising the step of: (iv) storing the frozen fragments at a temperature of at least less than -130°C.

23. 23. The method of claim 22, wherein the frozen fragments are stored in the vapor phase of liquid nitrogen.

24. 23. The method of claim 22, wherein the frozen sections are stored submerged in liquid nitrogen.

25. 1. A method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy, comprising: (a) storing tumor tissue in a frozen state, said method of storing tumor tissue comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) Freezing placing said tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and the cryopreservation medium at a temperature between 2 and 8° C. for 30 to 60 minutes; (iv) freezing the container, the freezing being a flash freezing using the vapor phase of liquid nitrogen; and (v) storing the container at a temperature of −130° C. or lower; Contains; (b) thawing the container; (c) treating said tumor tissue within a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 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. A method comprising:

26. 26. The method of claim 25, wherein the tumor tissue in step (i) is trimmed to a diameter of between 1.5 mm and 6 mm.

27. 27. The method of claim 26, wherein the tumor tissue in step (i) is trimmed to 6 mm x 6 mm x 6 mm.

28. The method of any one of claims 25 to 27, wherein the cryopreservation medium further comprises an antibacterial and antifungal agent.

29. 29. The method of any one of claims 25 to 28, wherein the cryopreservation medium comprises 2% v / v to 12% v / v dimethylsulfoxide (DMSO).

30. 30. The method of claim 29, wherein the cryopreservation medium comprises 10% DMSO.

31. The method of any one of claims 25 to 30, wherein the cryopreservation medium comprises gentamicin at a concentration of at least 50 μg / mL.

32. The method of any one of claims 25 to 31, wherein the tumor tissue is first washed with Hank's Balanced Salt Solution (HBSS) before carrying out step (i).

33. 33. The method of claim 32, wherein the washing comprises three successive washes of at least 3 minutes each, with the HBSS being replaced after each successive wash.

34. 34. The method of any one of claims 25 to 33, wherein step (b) comprises immersing the container in a water bath at a temperature of 37°C for 5 minutes.

35. 26. The method of claim 1 or claim 25, 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.

36. 26. The method of claim 25, wherein the method further comprises splitting between the first expansion step (c) and the second expansion step (e).

37. 37. The method of claim 36, wherein the splitting occurs 16 days after the initiation of step (c).

38. The method according to any one of claims 25 to 37, wherein the first culture step is for 11 days.

39. 38. The method of any one of claims 25 to 37, wherein steps (c) to (e) are carried out over a period of 22 days.

40. 38. The method of any one of claims 25 to 37, wherein steps (c) to (e) are carried out for a period of less than 20 days.

41. 26. The method of claim 25, wherein step (iv) comprises freezing the container at between -125°C and -150°C.

42. 26. The method of claim 25, wherein step (v) comprises storing the container at a temperature below -130°C.

43. 26. The method of claim 25, wherein step (v) comprises storing the container submerged in liquid nitrogen.

44. 1. A method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy, comprising: (a) storing the tumor tissue in a frozen state, the method comprising: (i) trimming the tumor tissue to remove excess non-tumor tissue; (ii) Freezing placing said tumor tissue in a closable container containing a storage medium; (iii) incubating the container containing the tumor tissue and the cryopreservation medium at 2-8° C. for 30 minutes; (iv) freezing the container using the vapor phase of liquid nitrogen; and (v) storing said container in the vapor phase at liquid nitrogen temperature. Contains; (b) thawing the container; (c) treating said tumor tissue within a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 to provide TILs; (d) removing at least some of said TILs; (e) expanding the TILs in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3, and IL-2 in a gas permeable container to provide an expanded number of TILs. A method comprising:

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