Processes for production of tumor infiltrating lymphocytes and uses of the same in immunotherapy

Abstract

To provide improved and / or shortened methods for expanding TILs and producing therapeutic populations of TILs.SOLUTION: Methods herein include novel methods for expanding TIL populations in a closed system that lead to improved efficacy, improved phenotype, and increased metabolic health of the TILs in a shorter time period, while allowing for reduced microbial contamination as well as decreased costs. Such TILs find use in therapeutic treatment regimens.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 775,954, filed on November 5, 2018, and U.S. and U.S. Provisional Patent Application No. 62 / 903,585, filed on September 20, 2019, which are hereby incorporated by reference in their entirety.

Background Art

[0002] The adoptive transfer of tumor-infiltrating lymphocytes (TILs) has emerged as a powerful approach for treating patients with poor prognoses suffering from large, refractory cancers. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Large numbers of TILs are required for successful immunotherapy, and a robust and reliable process is needed for commercialization. This has been difficult to achieve due to technical, logistical, and regulatory issues related to cell expansion. IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has become a preferred method of TIL expansion because of its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54, Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57, Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39, Riddell, et al., Science 1992, 257, 238-41, Dudley, et al., J. Immunother. 2003, 26, 332-42. The REP requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) from multiple donors as feeder cells, as well as anti-CD3 antibody (OKT3) and high-dose IL-2, but can result in a 1,000-fold expansion of TILs over 14 days. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs that have undergone the REP procedure have been successful in adoptive cell therapy following host immunosuppression in patients with melanoma. Current infusion parameters depend on readings of the TIL composition (e.g., CD28, CD8, or CD4 positive), as well as the fold expansion and viability of the REP product.

[0003] The current TIL manufacturing process is limited by concerns regarding duration, cost, sterility, and other factors described herein. There is an urgent need to provide a TIL manufacturing process and therapies based on such a process that feature improved cost-effectiveness and scalability in manufacturing, as well as a more potent anti-cancer phenotype of TIL formulations produced for the treatment of human patients at multiple clinical centers. The present invention provides a novel TIL expansion process that includes antigen-presenting feeder cells from the start of expansion to prime TIL for expansion, rather than the conventional REP pre-expansion step, thereby significantly shortening the overall time for the expansion process and fulfilling this need.

Summary of the Invention

[0004] The present invention provides improved and / or shortened methods for expanding TIL and producing a therapeutically effective TIL population.

[0005] The present invention is a method for expanding tumor-infiltrating lymphocytes (TIL) into a therapeutically effective TIL population, comprising (a) obtaining and / or receiving a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; and (b) performing a first priming expansion to produce a second TIL population by culturing the first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen-presenting cells (APC), wherein the first priming expansion is performed in a container comprising a first gas-permeable surface area and the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, the second TIL population being greater in number than the first TIL population. (c) culturing the second TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen-presenting cells (APC), wherein the second TIL population is cultured in a container comprising a second gas-permeable surface area and the second TIL population is cultured for a second period of about 1 to 7 / 8 days to obtain a third TIL population, the third TIL population being greater in number than the second TIL population. (c) Performing a second rapid expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APC to produce a third TIL population, wherein the number of APC added in the second rapid expansion is at least twice the number of APC added in step (b), the second rapid expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, the third TIL population is the therapeutic TIL population, and the second rapid expansion is performed in a container containing a second gas-permeable surface area, producing the third TIL population; (d) Harvesting the therapeutic TIL population obtained from step (c); (e) Transferring the harvested TIL population from step (d) to an infusion bag, providing a method comprising.

[0006] The present invention is a method for expanding tumor-infiltrating lymphocytes (TIL) into a therapeutic TIL population, (a) Obtaining and / or receiving a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Performing a first priming expansion to produce a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APC), wherein the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, and the second TIL population has a greater number than the first TIL population, producing the second TIL population; (c) Performing a second rapid expansion to produce a third TIL population by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3, and APC, wherein the second rapid expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, and the third TIL population is the therapeutic TIL population, producing the third TIL population; (d) Harvesting the therapeutic TIL population obtained from step (c), providing a method comprising.

[0007] In some embodiments, "obtaining" indicates that the TILs used in the method and / or process can be directly derived from a sample (including surgical resection, needle biopsy, core biopsy, fine biopsy, or other samples) as part of the method and / or process steps. In some embodiments, "receiving" indicates that the TILs used in the method and / or process are indirectly derived from a sample (including surgical resection, needle biopsy, core biopsy, fine biopsy, or other samples) and can then be used in the method and / or process (for example, if step (a) begins with TILs that have already been derived from the sample by another process not included in part (a), such TILs can be referred to as "received").

[0008] In some embodiments of the method, in step (b), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium of step (c) is greater than the number of APCs in the culture medium of step (b).

[0009] The present invention is a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutically effective TIL population, (a) performing a first priming expansion by culturing a first TIL population that can be obtained by treating a tumor sample from a tumor excised from a subject in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APCs) into a plurality of tumor fragments, wherein the first priming expansion is performed in a container having a first gas-permeable surface area for a first period of about 1 to 7 / 8 days to obtain a second TIL population, and the second TIL population is larger in number than the first TIL population, producing the second TIL population; (b) Performing a second rapid expansion by contacting the second TIL population with a cell culture medium of a second TIL population having additional IL-2, OKT-3, and APC to produce a third TIL population, wherein the number of APCs in the second rapid expansion is at least twice the number of APCs in step (a), the second rapid expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, the third TIL population is a therapeutic TIL population, and the second rapid expansion is performed in a container including a second gas permeable surface area, producing a third TIL population; (c) Collecting the therapeutic TIL population obtained from step (b). A method including the above steps is provided.

[0010] The present invention also provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, (a) Performing a first priming expansion by culturing a first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen presenting cells (APC) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain a second TIL population, and the number of the second TIL population is larger than that of the first TIL population, producing a second TIL population; (b) Performing a second rapid expansion by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 1 to 11 days to obtain a third TIL population, and the third TIL population is a therapeutic TIL population, producing a third TIL population; (c) Collecting the therapeutic TIL population obtained from step (b). A method including the above steps is provided.

[0011] In some embodiments of the present method, in step (a), the cell culture medium further contains antigen presenting cells (APC), and the number of APCs in the culture medium of step (c) is larger than the number of APCs in the culture medium of step (b).

[0012] In some embodiments, the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion is selected from the range of about 1.5:1 to 20:1.

[0013] In some embodiments, the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion is in the range of about 1.5:1 to 10:1.

[0014] In some embodiments, the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion is in the range of about 2:1 to 5:1.

[0015] In some embodiments, the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion is in the range of about 2:1 to 3:1.

[0016] In some embodiments, the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion is about 2:1.

[0017] In some embodiments, the number of APCs in the first priming expansion is about 1.0×10 6 APCs / cm 2 ~ about 4.5×10 6 APCs / cm 2 is selected from the range of, and the number of APCs in the second rapid expansion is about 2.5×10 6 APCs / cm 2 ~ about 7.5×10 6 APCs / cm 2 is selected from the range of.

[0018] In some embodiments, the number of APCs in the first priming expansion is about 1.5×10 6 APCs / cm 2 ~ about 3.5×10 6 APCs / cm 2 is selected from the range of, and the number of APCs in the second rapid expansion is about 3.5×10 6 APCs / cm2 ~ about 6.0×10 6 APC / cm 2 is selected from the range of.

[0019] In some embodiments, the number of APCs in the first priming expansion is about 2.0×10 6 APC / cm 2 ~ about 3.0×10 6 APC / cm 2 is selected from the range of, and the number of APCs in the second rapid expansion is about 4.0×10 6 APC / cm 2 ~ about 5.5×10 6 APC / cm 2 is selected from the range of.

[0020] In some embodiments, the number of APCs in the first priming expansion is about 1×10 8 APC ~ about 3.5×10 8 is selected from the range of APCs, and the number of APCs in the second rapid expansion is about 3.5×10 8 APC ~ about 1×10 9 is selected from the range of APCs.

[0021] In some embodiments, the number of APCs in the first priming expansion is about 1.5×10 8 APC ~ about 3×10 8 is selected from the range of APCs, and the number of APCs in the second rapid expansion is about 4×10 8 APC ~ about 7.5×10 8 is selected from the range of APCs.

[0022] In some embodiments, the number of APCs in the first priming expansion is about 2×10 8 APC ~ about 2.5×10 8 is selected from the range of APCs, and the number of APCs in the second rapid expansion is about 4.5×10 8 APC ~ about 5.5×10 8 is selected from the range of APCs.

[0023] In some embodiments, about 2.5×10 8The APC of 8 is added to the first priming expansion, and 5×10

[0024] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is from about 1.5:1 to about 100:1.

[0025] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 50:1.

[0026] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 25:1.

[0027] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 20:1.

[0028] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 10:1.

[0029] In some embodiments, the second TIL population is at least 50 times more numerous than the first TIL population.

[0030] In some embodiments, the method includes, after the step of harvesting a therapeutic TIL population, performing an additional step of transferring the harvested therapeutic TIL population to an infusion bag.

[0031] In some embodiments, a plurality of tumor fragments are distributed among a plurality of separate containers, and in each of the separate containers, a second TIL population is obtained from the first TIL population in a first priming expansion step, a third TIL population is obtained from the second TIL population in a second rapid expansion step, and a therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to yield a harvested TIL population.

[0032] In some embodiments, the plurality of separate containers includes at least two separate containers.

[0033] In some embodiments, the plurality of separate containers includes from 2 to 20 separate containers.

[0034] In some embodiments, the plurality of separate containers includes from 2 to 10 separate containers.

[0035] In some embodiments, the plurality of separate containers includes from 2 to 5 separate containers.

[0036] In some embodiments, each of the separate containers includes a first gas-permeable surface area.

[0037] In some embodiments, the plurality of tumor fragments are distributed among a single container.

[0038] In some embodiments, the single container includes a first gas-permeable surface area.

[0039] In some embodiments, in the first priming expansion step, the cell culture medium includes antigen-presenting cells (APCs), and the APCs are layered on the first gas-permeable surface area with an average thickness of from about 1 cell layer to about 3 cell layers.

[0040] In some embodiments, in the first priming expansion step, the APCs are layered on the first gas-permeable surface area with an average thickness of from about 1.5 cell layers to about 2.5 cell layers.

[0041] In some embodiments, in the step of the first priming expansion, the APCs are layered on the first gas permeable surface area with an average thickness of about two cell layers.

[0042] In some embodiments, in the step of the second rapid expansion, the APCs are layered on the first gas permeable surface area with a thickness of about three to about five cell layers.

[0043] In some embodiments, the APCs in the second rapid expansion are layered on the first gas permeable surface area with a thickness of about 3.5 to about 4.5 cell layers.

[0044] In some embodiments, the APCs in the second rapid expansion are layered on the first gas permeable surface area with a thickness of about four cell layers.

[0045] In some embodiments, in the step of the first priming expansion, the first priming expansion is performed in a first container containing the first gas permeable surface area, and in the step of the second rapid expansion, the second rapid expansion is performed in a second container containing the second gas permeable surface area.

[0046] In some embodiments, the second container is larger than the first container.

[0047] In some embodiments, in the step of the first priming expansion, the cell culture medium contains antigen-presenting cells (APCs), and the APCs are layered on the first gas permeable surface area with an average thickness of about one to about three cell layers. on the first gas permeable surface area.

[0048] In some embodiments, in the step of the first priming expansion, the APCs are layered on the first gas permeable surface area with an average thickness of about 1.5 to about 2.5 cell layers.

[0049] In some embodiments, in the step of the first priming expansion, the APCs are layered on the first gas-permeable surface area with an average thickness of about two cell layers.

[0050] In some embodiments, the APCs of the second rapid expansion are layered on the second gas-permeable surface area with an average thickness of about three to about five cell layers.

[0051] In some embodiments, the APCs of the second rapid expansion are layered on the second gas-permeable surface area with an average thickness of about 3.5 to about 4.5 cell layers.

[0052] In some embodiments, in the step of the second rapid expansion, the APCs are layered on the second gas-permeable surface area with an average thickness of about four cell layers.

[0053] In some embodiments, for each container in which the first priming expansion is performed on the first TIL population, the second rapid expansion is performed on the second TIL population generated from such first TIL population within the same container.

[0054] In some embodiments, each container includes a first gas-permeable surface area.

[0055] In some embodiments, in the step of the first priming expansion, the cell culture medium contains antigen-presenting cells (APCs), and the APCs are layered on the first gas-permeable surface area with an average thickness of about one to about three cell layers.

[0056] In some embodiments, in the step of the first priming expansion, the APCs are layered on the first gas-permeable surface area with an average thickness of about 1.5 to about 2.5 cell layers.

[0057] In some embodiments, in the step of the first priming expansion, the APCs are layered on the first gas-permeable surface area with an average thickness of about two cell layers.

[0058] In some embodiments, in the second rapid expansion step, the APCs are layered on the first gas permeable surface area with an average thickness of about 3 cell layers to about 5 cell layers.

[0059] In some embodiments, in the second rapid expansion step, the APCs are layered on the first gas permeable surface area with an average thickness of about 3.5 cell layers to about 4.5 cell layers.

[0060] In some embodiments, in the second rapid expansion step, the APCs are layered on the first gas permeable surface area with an average thickness of about 4 cell layers.

[0061] In some embodiments, for each container in which the first priming expansion is performed on the first TIL population in the first priming expansion step, the first container includes a first surface area, the cell culture medium includes antigen presenting cells (APCs), the APCs are layered on the first gas permeable surface area, and the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step is selected from the range of about 1:1.1 to about 1:10.

[0062] In some embodiments, the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step is selected from the range of about 1:1.2 to about 1:8.

[0063] In some embodiments, the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step is selected from the range of about 1:1.3 to about 1:7.

[0064] In some embodiments, the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step is selected from the range of about 1:1.4 to about 1:6.

[0065] In some embodiments, the ratio of the average number of layers of APC stratified in the first priming expansion step to the average number of layers of APC stratified in the second rapid expansion step is selected from the range of about 1:1.5 to about 1:5.

[0066] In some embodiments, the ratio of the average number of layers of APC stratified in the first priming expansion step to the average number of layers of APC stratified in the second rapid expansion step is selected from the range of about 1:1.6 to about 1:4.

[0067] In some embodiments, the ratio of the average number of layers of APC stratified in the first priming expansion step to the average number of layers of APC stratified in the second rapid expansion step is selected from the range of about 1:1.7 to about 1:3.5.

[0068] In some embodiments, the ratio of the average number of layers of APC stratified in the first priming expansion step to the average number of layers of APC stratified in the second rapid expansion step is selected from the range of about 1:1.8 to about 1:3.

[0069] In some embodiments, the ratio of the average number of layers of APC stratified in the first priming expansion step to the average number of layers of APC stratified in the second rapid expansion step is selected from the range of about 1:1.9 to about 1:2.5.

[0070] In some embodiments, the ratio of the average number of layers of APC stratified in the first priming expansion step to the average number of layers of APC stratified in the second rapid expansion step is about 1:2.

[0071] In some embodiments, after the second rapid expansion step of 2 to 3 days, additional IL-2 is supplemented to the cell culture medium.

[0072] In some embodiments, the method further comprises cryopreserving the collected TIL population in the step of collecting the therapeutic TIL population using a cryopreservation process.

[0073] In some embodiments, the method further comprises the step of cryopreserving the infusion bag.

[0074] In some embodiments, the cryopreservation process is performed using the collected TIL population and cryopreservation medium at a 1:1 ratio.

[0075] In some embodiments, the antigen-presenting cell is a peripheral blood mononuclear cell (PBMC).

[0076] In some embodiments, the PBMCs are irradiated and allogeneic.

[0077] In some embodiments, in the step of first priming expansion, the cell culture medium contains peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the step of first priming expansion is about 2.5×10 8 is.

[0078] In some embodiments, in the step of second rapid expansion, the antigen-presenting cells (APCs) in the cell culture medium are peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the step of second rapid expansion is about 5×10 8 is.

[0079] In some embodiments, the antigen-presenting cell is an artificial antigen-presenting cell.

[0080] In some embodiments, the collection in the step of collecting the therapeutic TIL population is performed using a membrane-based cell processing system.

[0081] In some embodiments, the collection in the step of collecting the therapeutic TIL population is performed using a LOVO cell processing system.

[0082] In some embodiments, the plurality of fragments includes about 60 fragments per container in the first priming extension step, and each fragment has a volume of about 27 mm 3 .

[0083] In some embodiments, the plurality of fragments includes about 30 to about 60 fragments, and the total volume is about 1300 mm 3 to about 1500 mm 3 .

[0084] In some embodiments, the plurality of fragments includes about 50 fragments, and the total volume is about 1350 mm 3 .

[0085] In some embodiments, the plurality of fragments includes about 50 fragments, and the total mass is about 1 gram to about 1.5 grams.

[0086] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell bags.

[0087] In some embodiments, the IL-2 concentration is about 10,000 IU / mL to about 5,000 IU / mL.

[0088] In some embodiments, the IL-2 concentration is about 6,000 IU / mL.

[0089] In some embodiments, the infusion bag in the step of transferring the collected therapeutic TIL population to the infusion bag is an infusion bag containing HypoThermosol.

[0090] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO).

[0091] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0092] In some embodiments, the first period of the first priming expansion step and the second period of the second rapid expansion step are each performed individually within a period of 5, 6, or 7 days.

[0093] In some embodiments, the first period of the first priming expansion step is performed within a period of 5, 6, or 7 days.

[0094] In some embodiments, the second period of the second rapid expansion step is performed within a period of 7, 8, or 9 days.

[0095] In some embodiments, the first period of the first priming expansion step and the second period of the second rapid expansion step are each performed individually within a period of 7 days.

[0096] In some embodiments, the first priming expansion step following collection of the therapeutic TIL population is performed within a period of about 14 days to about 16 days.

[0097] In some embodiments, the first priming expansion step following collection of the therapeutic TIL population is performed within a period of about 15 days to about 16 days.

[0098] In some embodiments, the first priming expansion step following collection of the therapeutic TIL population is performed within a period of about 14 days.

[0099] In some embodiments, the first priming expansion step following collection of the therapeutic TIL population is performed within a period of about 15 days.

[0100] In some embodiments, the first priming expansion step following collection of the therapeutic TIL population is performed within a period of about 16 days.

[0101] In some embodiments, the method further comprises cryopreserving the collected therapeutic TIL population using a cryopreservation process, and the steps of the first priming expansion through the collection of the therapeutic TIL population and the cryopreservation step are performed within 16 days or less.

[0102] In some embodiments, the therapeutic TIL population collected in the step of collecting the therapeutic TIL population contains sufficient TIL for a therapeutically effective dose of TIL.

[0103] In some embodiments, the number of TIL sufficient for a therapeutically effective dose is about 2.3×10 10 ~ about 13.7×10 10 .

[0104] In some embodiments, the third TIL population in the second rapid expansion step provides increased efficacy, increased interferon-γ production, and / or increased polyclonality.

[0105] In some embodiments, the third TIL population in the second rapid expansion step provides at least 1 to 5 times or more interferon-γ production compared to TIL prepared by a process of 18 days or more.

[0106] In some embodiments, the effector T cells and / or central memory T cells obtained from the third TIL population in the second rapid expansion step exhibit increased CD8 and CD28 expression compared to the effector T cells and / or central memory T cells obtained from the second TIL population in the first priming expansion step.

[0107] In some embodiments, the therapeutic TIL population from the step of collecting the therapeutic TIL population is infused into the patient.

[0108] The present invention also provides a method for treating a subject having cancer, (a) Obtaining and / or receiving a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Performing a first priming expansion to produce a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, optionally OKT-3, and optionally antigen-presenting cells (APC), wherein the first priming expansion is performed in a container containing a first gas-permeable surface area, the first priming expansion is performed for about 1 to 7 / 8 days, a second TIL population is obtained, and the second TIL population is at least 50 times more numerous than the first TIL population; (c) Performing a second rapid expansion to produce a third TIL population by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APC, wherein the number of APCs added for the second rapid expansion is at least twice the number of APCs added in step (b), the second rapid expansion is performed for about 1 to 11 days, a third TIL population is obtained, the third TIL population is a therapeutic TIL population, and the second rapid expansion is performed in a container containing a second gas-permeable surface area; (d) Harvesting the therapeutic TIL population obtained from step (c); (e) Transferring the harvested TIL population from step (d) to an infusion bag; (f) Administering a therapeutically effective dose of the TIL population from step (e) to the subject. Provided is a method comprising administering expanded tumor-infiltrating lymphocytes (TIL).

[0109] In some embodiments, the number of TILs sufficient to administer a therapeutically effective dose in step (f) is about 2.3×10 10 ~ about 13.7×10 10 .

[0110] In some embodiments, the antigen-presenting cells (APC) are PBMC.

[0111] In some embodiments, a non-myeloablative lymphodepletion regimen is administered to the patient prior to administering a therapeutically effective dose of TIL cells in step (f).

[0112] In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m 2 / day for 2 days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for 5 days.

[0113] In some embodiments, the method further comprises treating the patient with a high-dose IL-2 regimen that begins on the day following administration of the TIL cells to the patient in step (f).

[0114] In some embodiments, the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours up to the tolerated limit.

[0115] In some embodiments, the third TIL population in step (b) provides increased efficacy, increased interferon-γ production, and / or increased polyclonality.

[0116] In some embodiments, the third TIL population in step (c) provides at least 1 to 5-fold or more interferon-γ production compared to TIL prepared by a process of 16 days or more.

[0117] In some embodiments, the effector T cells and / or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population in step (b).

[0118] In some embodiments, the cancer is a solid tumor.

[0119] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0120] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0121] In some embodiments, the cancer is melanoma.

[0122] In some embodiments, the cancer is HNSCC.

[0123] In some embodiments, the cancer is cervical cancer.

[0124] In some embodiments, the cancer is NSCLC.

[0125] In some embodiments, the cancer is glioblastoma (including GBM).

[0126] In some embodiments, the cancer is gastrointestinal cancer.

[0127] In some embodiments, the cancer is a high-frequency mutant cancer.

[0128] In some embodiments, the cancer is a pediatric high-frequency mutant cancer.

[0129] In some embodiments, the container is a sealed container.

[0130] In some embodiments, the container is a G container.

[0131] In some embodiments, the container is GREX-10.

[0132] In some embodiments, the sealed container contains GREX-100.

[0133] In some embodiments, the sealed container contains GREX-500.

[0134] The present invention also provides a therapeutic population of tumor infiltrating lymphocytes (TILs) produced by the methods disclosed herein.

[0135] The present invention also provides a therapeutic population of tumor infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue that provides increased efficacy, increased interferon gamma production, and / or increased polyclonality.

[0136] In some embodiments, the therapeutic TIL population disclosed herein provides increased interferon gamma production.

[0137] In some embodiments, the therapeutic TIL population disclosed herein provides increased polyclonality.

[0138] In some embodiments, the therapeutic TIL population disclosed herein provides increased efficacy.

[0139] In some embodiments, the therapeutic TIL population described herein is capable of producing at least one-fold more interferon gamma compared to TILs prepared by a process of 16 days or more.

[0140] In some embodiments, the therapeutic TIL population described herein is capable of producing at least two-fold more interferon gamma compared to TILs prepared by a process of 16 days or more.

[0141] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three-fold more interferon gamma compared to TILs prepared by a process of 16 days or more.

[0142] In some embodiments, the present invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) that are capable of producing at least one-fold more interferon-γ as compared to TILs prepared by a process in which a first TIL expansion is performed without adding any antigen-presenting cells (APCs).

[0143] In some embodiments, the therapeutic TIL population described herein is capable of producing at least two-fold more interferon-γ as compared to TILs prepared by a process in which a first TIL expansion is performed without adding any APCs.

[0144] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three-fold more interferon-γ as compared to TILs prepared by a process in which a first TIL expansion is performed without adding any APCs.

[0145] In some embodiments, the present invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) that are capable of producing at least one-fold more interferon-γ as compared to TILs prepared by a process in which a first TIL expansion is performed without adding any OKT3.

[0146] In some embodiments, the therapeutic TIL population described herein is capable of producing at least two-fold more interferon-γ as compared to TILs prepared by a process in which a first TIL expansion is performed without adding any OKT3.

[0147] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three-fold more interferon-γ as compared to TILs prepared by a process in which a first TIL expansion is performed without adding any OKT3.

[0148] In some embodiments, the present invention provides a population of therapeutic tumor-infiltrating lymphocytes (TILs) that are capable of producing at least one-fold more interferon-γ compared to TILs prepared by a process in which a first TIL expansion is performed without adding any antigen-presenting cells (APCs) or any OKT3.

[0149] In some embodiments, the therapeutic TIL population described herein is capable of producing at least two-fold more interferon-γ compared to TILs prepared by a process in which a first TIL expansion is performed without adding any antigen-presenting cells (APCs) or any OKT3.

[0150] In some embodiments, the therapeutic TIL population described herein is capable of producing at least three-fold more interferon-γ compared to TILs prepared by a process in which a first TIL expansion is performed without adding any antigen-presenting cells (APCs) or any OKT3.

[0151] The present invention also provides a tumor-infiltrating lymphocyte (TIL) composition comprising the therapeutic TIL population described herein and a pharmaceutically acceptable carrier.

[0152] The present invention also provides a sterile infusion bag containing the TIL composition described herein.

[0153] The present invention also provides a cryopreserved preparation of a therapeutic TIL population as described herein.

[0154] The present invention also provides a tumor-infiltrating lymphocyte (TIL) composition comprising the therapeutic TIL population described herein and a cryopreservation medium.

[0155] In some embodiments, the cryopreservation medium contains DMSO.

[0156] In some embodiments, the cryopreservation medium contains 7 - 10% DMSO.

[0157] The present invention also provides a cryopreserved preparation of the TIL composition described herein.

[0158] In some embodiments, the tumor-infiltrating lymphocyte (TIL) composition described herein is for use as a medicament.

[0159] In some embodiments, the tumor-infiltrating lymphocyte (TIL) composition described herein is for use in the treatment of cancer.

[0160] In some embodiments, the tumor-infiltrating lymphocyte (TIL) composition described herein is for use in the treatment of solid tumor cancers.

[0161] In some embodiments, the tumor-infiltrating lymphocyte (TIL) composition described herein is for use in the treatment of cancer selected from melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0162] In some embodiments, the tumor-infiltrating lymphocyte (TIL) composition described herein is for use in the treatment of cancer selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0163] In some embodiments, the TIL composition described herein is for use in the treatment of cancer, wherein the cancer is melanoma.

[0164] In some embodiments, the TIL composition described herein is for use in the treatment of cancer, wherein the cancer is HNSCC.

[0165] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is cervical cancer.

[0166] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is NSCLC.

[0167] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is glioblastoma (including GBM).

[0168] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is gastrointestinal cancer.

[0169] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is highly mutated cancer.

[0170] In some embodiments, the TIL compositions described herein are for use in the treatment of cancer, which is pediatric highly mutated cancer.

[0171] In some embodiments, the present invention provides the use of a tumor-infiltrating lymphocyte (TIL) composition described herein in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the TIL composition. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is HNSCC. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is glioblastoma (including GBM). In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is a high-frequency mutant cancer. In some embodiments, the cancer is a pediatric high-frequency mutant cancer.

[0172] In some embodiments, the tumor-infiltrating lymphocyte (TIL) composition described herein is for use in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the TIL composition. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0173] The present invention also provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of the tumor-infiltrating lymphocyte (TIL) composition described herein.

[0174] In some embodiments, the cancer is a solid tumor.

[0175] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSC LC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0176] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is HNSCC. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is glioblastoma (including GBM). In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is a high-frequency mutated cancer. In some embodiments, the cancer is a pediatric high-frequency mutated cancer.

[0177] The present invention also provides a method of expanding T cells, comprising: (a) performing a first priming expansion of a first T cell population obtained from a donor by culturing the first T cell population to effect growth and priming the activation of the first T cell population; (b) after the activation of the first T cell population primed in step (a) begins to decline, performing a second rapid expansion of the first T cell population by culturing the first T cell population to effect growth and promoting the activation of the first T cell population to obtain a second T cell population; (c) harvesting the second T cell population.

[0178] In some embodiments, the first priming expansion of step (a) is performed over a period of up to 7 days.

[0179] In some embodiments, the second rapid expansion of step (b) is performed over a period of up to 11 days.

[0180] In some embodiments, the second rapid expansion of step (b) is performed over a period of up to 9 days.

[0181] In some embodiments, the first priming expansion of step (a) is performed over a 7-day period, and the second rapid expansion of step (b) is performed over a 9-day period.

[0182] In some embodiments, the first priming expansion of step (a) is performed over a period of up to 8 days.

[0183] In some embodiments, the second rapid expansion of step (b) is performed over a period of up to 8 days.

[0184] In some embodiments, the first priming expansion of step (a) is performed over an 8-day period, and the second rapid expansion of step (b) is performed over an 8-day period.

[0185] In some embodiments of the method, in step (a), the first T cell population is cultured in a first culture medium containing OKT-3 and IL-2.

[0186] In some embodiments, the first culture medium contains OKT-3, IL-2, and antigen-presenting cells (APCs).

[0187] In some embodiments of the method, in step (b), the first T cell population is cultured in a second culture medium containing OKT-3, IL-2, and antigen-presenting cells (APCs).

[0188] In some embodiments of the method, in step (a), the first T cell population is cultured in a first culture medium in a container comprising a first gas-permeable surface, the first culture medium optionally comprising OKT-3, IL-2, and optionally a first antigen-presenting cell (APC) population, the first APC population being exogenous to the donor of the first T cell population, the first APC population being layered on the first gas-permeable surface, and in step (b), the first T cell population is cultured in a second culture medium in the container, the second culture medium comprising OKT-3, IL-2, and a second APC population, the second APC population being exogenous to the donor of the first T cell population, the second APC population being layered on the first gas-permeable surface, and the second APC population being larger than the first APC population.

[0189] In some embodiments, the ratio of the number of APCs in the second APC population to the number of APCs in the first APC population is about 2:1.

[0190] In some embodiments, the number of APCs in the first APC population is about 2.5×10 8 and the number of APCs in the second APC population is about 5×10 8

[0191] In some embodiments, in step (a), the first APC population is layered on the first gas-permeable surface with an average thickness of two layers of APCs.

[0192] In some embodiments, in step (b), the second APC population is layered on the first gas-permeable surface with an average thickness selected from the range of 4 to 8 layers of APCs.

[0193] In some embodiments, the ratio of the average number of layers of APCs layered on the first gas-permeable surface in step (b) to the average number of layers of APCs layered on the first gas-permeable surface in step (a) is 2:1.

[0194] ​In some embodiments, the APCs are peripheral blood mononuclear cells (PBMCs).

[0195] In some embodiments, the APCs include PBMCs that are irradiated and exogenous to the donor of the first T cell population.

[0196] In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs).

[0197] In some embodiments, the T cells are marrow infiltrating lymphocytes (MILs).

[0198] In some embodiments, the T cells are peripheral blood lymphocytes (PBLs).

[0199] In some embodiments, the cell culture medium is a synthetic medium and / or a serum-free medium.

[0200] In some embodiments, the synthetic medium includes (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0201] In some embodiments, the serum-free or synthetic medium includes a basal cell medium as well as a serum supplement and / or a serum replacement.

[0202] In some embodiments, the basal cell medium includes, but is not limited to, CTS(™) OpTmizer(™) T-cell Expansion Basal Medium, CTS(™) OpTmizer(™) T-Cell Expansion SFM, CTS(™) AIM-V Medium, CTS(™) AIM-V SFM, LymphoONE(™) T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0203] In some embodiments, the serum supplement or serum replacement is selected from CTS(™) OpTmizer T-Cell Expansion Serum Supplement and CTS(™) Immune Cell Serum Replacement.

[0204] In some embodiments, the cell culture medium includes one or more albumins or albumin substitutes.

[0205] In some embodiments, the cell culture medium includes one or more amino acids.

[0206] In some embodiments, the cell culture medium includes one or more vitamins, one or more transferrins or transferrin substitutes.

[0207] In some embodiments, the cell culture medium includes one or more antioxidants, one or more insulins or insulin substitutes.

[0208] In some embodiments, the cell culture medium includes one or more collagen precursors, one or more antibiotics, and one or more trace elements.

[0209] In some embodiments, the cell culture medium contains albumin.

[0210] In some embodiments, the cell culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and a trace element moiety Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3” , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ and a compound containing the same.

[0211] In some embodiments, the cell culture medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0212] In some embodiments, the total serum replacement concentration (volume %) of the cell culture medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the cell culture medium.

[0213] In some embodiments, the total serum replacement concentration of the cell culture medium is about 3%, about 5%, or about 10% of the total volume of the cell culture medium.

[0214] In some embodiments, the cell culture medium further comprises glutamine (i.e., GlutaMAX™) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM.

[0215] In some embodiments, the cell culture medium further comprises glutamine (i.e., GlutaMAX™) at a concentration of about 2 mM.

[0216] In some embodiments, the cell culture medium further comprises 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM.

[0217] In some embodiments, the cell culture medium further comprises 2-mercaptoethanol at a concentration of about 55 mM.

[0218] In some embodiments, the cell culture medium comprises a synthetic medium as described in International PCT Publication No. WO / 1998 / 030679.

[0219] In some embodiments, the cell culture medium comprises glycine in the range of about 5 to 200 mg / L, L-histidine in the range of about 5 to 250 mg / L, L-isoleucine in the range of about 5 to 300 mg / L, L-methionine in the range of about 5 to 200 mg / L, L-phenylalanine in the range of about 5 to 400 mg / L, L-proline in the range of about 1 to 1000 mg / L, L-hydroxyproline in the range of about 1 to 45 mg / L, L-serine in the range of about 1 to 250 mg / L, L-threonine in the range of about 10 to 500 mg / L, L-tryptophan in the range of about 2 to 110 mg / L, L-tyrosine in the range of about 3 to 175 mg / L, L-valine in the range of about 5 to 500 mg / L, thiamine in the range of about 1 to 20 mg / L, reduced glutathione in the range of about 1 to 20 mg / L, L-ascorbic acid-2-phosphate in the range of about 1 to 200 mg / L, iron-saturated transferrin in the range of about 1 to 50 mg / L, insulin in the range of about 1 to 100 mg / L, sodium selenite in the range of about 0.000001 to 0.0001 mg / L, and / or albumin (e.g., AlbuMAX® I) in the range of about 5000 to 50,000 mg / L.

[0220] In some embodiments, the cell culture medium comprises one or more of the non-trace element partial components in the synthetic medium, which are present in the concentration ranges listed in the column under the heading "Concentration Range in 1× Medium" in Table A provided herein.

[0221] In some embodiments, the osmotic pressure of the cell culture medium is about 260 to 350 mOsmol.

[0222] In some embodiments, the cell culture medium further comprises sodium bicarbonate at about 3.7 g / L, or about 2.2 g / L.

[0223] In some embodiments, the cell culture medium further comprises L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration about 100 μM), and / or 2-mercaptoethanol (final concentration about 100 μM).

[0224] In some embodiments, the cell culture medium in the first and / or second gas permeable containers lacks β-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

[0225] In some embodiments, the cell culture medium is CTS OpTmizer T-Cell Expansion SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

[0226] In some embodiments, the cell culture medium is CTS™ OpTmizer™ T-Cell Expansion Supplement (26 mL / L), and 3% CTS™ Immune Cell SR, and 2 mM Glutamax-supplemented CTS™ OpTmizer™ T-Cell Expansion Basal Medium, and optionally further contains 6,000 IU / mL of IL-2.

[0227] In some embodiments, the cell culture medium is CTS™ OpTmizer™ T-Cell Expansion Supplement (26 mL / L), and 3% CTS™ Immune Cell SR, 2 mM Glutamax-supplemented CTS™ OpTmizer™ T-Cell Expansion Basal Medium, and optionally further contains 3,000 IU / mL of IL-2.

[0228] The present invention also provides a tumor-infiltrating lymphocyte (TIL) composition comprising i) a population of therapeutic tumor-infiltrating lymphocytes (TIL), and ii) a synthetic or serum-free medium optionally containing (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0229] The present invention also provides an expanded tumor-infiltrating lymphocyte (TIL) composition comprising: i) a therapeutic population of tumor-infiltrating lymphocytes (TILs); and ii) a synthetic or serum-free medium optionally comprising (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0230] In some embodiments, the synthetic or serum-free medium comprises (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0231] In some embodiments, the synthetic or serum-free medium comprises a basal cell medium and a serum supplement and / or a serum replacement.

[0232] In some embodiments, the basal cell medium includes, but is not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco Medium.

[0233] In some embodiments, the serum supplement or serum replacement is selected from CTS™ OpTmizer T-Cell Expansion Serum Supplement and CTS™ Immune Cell Serum Replacement.

[0234] In some embodiments, the synthetic medium or serum-free medium contains one or more albumins or albumin substitutes.

[0235] In some embodiments, the synthetic medium or serum-free medium contains one or more amino acids.

[0236] In some embodiments, the synthetic medium or serum-free medium contains one or more vitamins, one or more transferrins or transferrin substitutes.

[0237] In some embodiments, the synthetic medium or serum-free medium contains one or more antioxidants, one or more insulins or insulin substitutes.

[0238] In some embodiments, the synthetic medium or serum-free medium contains one or more collagen precursors, one or more antibiotics, and one or more trace elements.

[0239] In some embodiments, the synthetic medium or serum-free medium contains albumin.

[0240] In some embodiments, the synthetic medium or serum-free medium contains albumin, and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element portion Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3” , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn2+ and Zr 4+ and one or more components selected from the group consisting of compounds containing

[0241] In some embodiments, the synthetic medium or serum-free medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0242] In some embodiments, the synthetic medium or serum-free medium has a total serum replacement concentration (volume %) of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the cell culture medium.

[0243] In some embodiments, the synthetic medium or serum-free medium has a total serum replacement concentration of about 3%, about 5%, or about 10% of the total volume of the cell culture medium.

[0244] In some embodiments, the synthetic medium or serum-free medium further comprises glutamine (i.e., GlutaMAX™) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM.

[0245] In some embodiments, the synthetic medium or serum-free medium further comprises glutamine (i.e., GlutaMAX™) at a concentration of about 2 mM.

[0246] In some embodiments, the synthetic medium or serum-free medium further comprises 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM.

[0247] In some embodiments, the synthetic medium or serum-free medium further contains 2-mercaptoethanol at a concentration of about 55 mM.

[0248] In some embodiments, the synthetic medium or serum-free medium contains the synthetic medium described in International PCT Publication No. WO / 1998 / 030679.

[0249] In some embodiments, the synthetic medium or serum-free medium contains glycine in the range of about 5 - 200 mg / L, L-histidine in the range of about 5 - 250 mg / L, L-isoleucine in the range of about 5 - 300 mg / L, L-methionine in the range of about 5 - 200 mg / L, L-phenylalanine in the range of about 5 - 400 mg / L, L-proline in the range of about 1 - 1000 mg / L, L-hydroxyproline in the range of about 1 - 45 mg / L, L-serine in the range of about 1 - 250 mg / L, L-threonine in the range of about 10 - 500 mg / L, L-tryptophan in the range of about 2 - 110 mg / L, L-tyrosine in the range of about 3 - 175 mg / L, L-valine in the range of about 5 - 500 mg / L, thiamine in the range of about 1 - 20 mg / L, reduced glutathione in the range of about 1 - 20 mg / L, L-ascorbic acid-2-phosphate in the range of about 1 - 200 mg / L, iron-saturated transferrin in the range of about 1 - 50 mg / L, insulin in the range of about 1 - 100 mg / L, sodium selenite in the range of about 0.000001 - 0.0001 mg / L, and / or albumin (e.g., AlbuMAX® I) in the range of about 5000 - 50,000 mg / L.

[0250] In some embodiments, the synthetic medium or serum-free medium contains one or more of the non-trace element partial components in the synthetic medium, which are present in the concentration ranges listed in the columns under the heading "Concentration Range in 1× Medium" in Table A provided herein.

[0251] In some embodiments, the osmotic pressure of the synthetic medium or serum-free medium is about 260 - 350 mOsmol.

[0252] In some embodiments, the synthetic medium or serum-free medium further comprises sodium bicarbonate at about 3.7 g / L, or about 2.2 g / L.

[0253] In some embodiments, the synthetic medium or serum-free medium further comprises L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration about 100 μM), and / or 2-mercaptoethanol (final concentration about 100 μM).

[0254] In some embodiments, the synthetic medium or serum-free medium in the first and / or second gas-permeable container is lacking β-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

[0255] In some embodiments, the cell culture medium is CTS OpTmizer T-Cell Expansion SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

[0256] In some embodiments, the cell culture medium is CTS (trademark) OpTmizer (trademark) T-Cell Expansion Supplement (26 mL / L), and 3% CTS (trademark) Immune Cell SR, and 2 mM Glutamax-supplemented CTS (trademark) OpTmizer (trademark) T-Cell Expansion Basal Medium, and optionally further comprises 6,000 IU / mL of IL-2.

[0257] In some embodiments, the cell culture medium comprises CTS(™) OpTmizer(™) T-Cell Expansion Basal Medium supplemented with CTS(™) OpTmizer(™) T-Cell Expansion Supplement (26 mL / L), and 3% CTS(™) Immune Cell SR, 2 mM Glutamax, and optionally further comprises 3,000 IU / mL of IL-2.

[0258] In some embodiments, the TIL population is a therapeutic TIL population.

[0259] In some embodiments, the therapeutic TIL population shows an increase in serum IFN-γ, and the increase in IFN-γ is greater than 200 pg / ml, greater than 250 pg / ml, greater than 300 pg / ml, greater than 350 pg / ml, greater than 400 pg / ml, greater than 450 pg / ml, greater than 500 pg / ml, greater than 550 pg / ml, greater than 600 pg / ml, greater than 650 pg / ml, greater than 700 pg / ml, greater than 750 pg / ml, greater than 800 pg / ml, greater than 850 pg / ml, greater than 900 pg / ml, greater than 950 pg / ml, or greater than 1000 pg / ml.

Brief Description of the Drawings

[0260]

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[0261] Brief description of the sequence listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0262] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0263] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0264] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0265] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0266] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0267] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0268] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0269] SEQ ID NO: 9 is the amino acid sequence of human 4-1BB.

[0270] SEQ ID NO: 10 is the amino acid sequence of mouse 4-1BB.

[0271] SEQ ID NO: 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0272] SEQ ID NO: 12 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0273] SEQ ID NO: 13 is the variable heavy chain (VH) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0274] SEQ ID NO: 14 is the variable light chain (VL) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0275] SEQ ID NO: 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0276] SEQ ID NO: 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0277] SEQ ID NO: 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0278] Sequence number 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0279] Sequence number 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0280] Sequence number 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0281] Sequence number 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0282] Sequence number 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0283] Sequence number 23 is the heavy chain variable region (V H ) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0284] Sequence number 24 is the light chain variable region (V L ) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0285] Sequence number 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0286] Sequence number 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-66 3513).

[0287] Sequence number 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0288] Sequence number 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0289] Sequence number 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0290] Sequence number 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0291] Sequence number 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0292] Sequence number 32 is the linker of the TNFRSF agonist fusion protein.

[0293] Sequence number 33 is the linker of the TNFRSF agonist fusion protein.

[0294] Sequence number 34 is the linker of the TNFRSF agonist fusion protein.

[0295] Sequence number 35 is the linker of the TNFRSF agonist fusion protein.

[0296] Sequence number 36 is the linker of the TNFRSF agonist fusion protein.

[0297] Sequence number 37 is the linker of the TNFRSF agonist fusion protein.

[0298] Sequence number 38 is the linker of the TNFRSF agonist fusion protein.

[0299] Sequence number 39 is the linker of the TNFRSF agonist fusion protein.

[0300] Sequence number 40 is the linker of the TNFRSF agonist fusion protein.

[0301] Sequence number 41 is the linker of the TNFRSF agonist fusion protein.

[0302] Sequence number 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0303] Sequence number 43 is the linker of the TNFRSF agonist fusion protein.

[0304] Sequence number 44 is the linker of the TNFRSF agonist fusion protein.

[0305] Sequence number 45 is the linker of the TNFRSF agonist fusion protein.

[0306] Sequence number 46 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0307] Sequence number 47 is the soluble part of the 4-1BBL polypeptide.

[0308] Sequence number 48 is the heavy chain variable region (V H ) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0309] Sequence number 49 is the light chain variable region (V L ) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0310] Sequence number 50 is the heavy chain variable region (V H ) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0311] Sequence number 51 is the light chain variable region (V L ) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0312] Sequence number 52 is the heavy chain variable region (V H ) of the 4-1BB agonist antibody H39E3-2.

[0313] Sequence number 53 is the variable region of the light chain (V L ) of the 4-1BB agonist antibody H39E3-2.

[0314] Sequence number 54 is the amino acid sequence of human OX40.

[0315] Sequence number 55 is the amino acid sequence of mouse OX40.

[0316] Sequence number 56 is the heavy chain of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0317] Sequence number 57 is the light chain of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0318] Sequence number 58 is the variable region of the heavy chain (V H ) of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0319] Sequence number 59 is the variable region of the light chain (V L ) of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0320] Sequence number 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0321] Sequence number 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0322] Sequence number 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0323] Sequence number 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0324] SEQ ID NO: 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0325] SEQ ID NO: 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0326] SEQ ID NO: 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0327] SEQ ID NO: 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0328] SEQ ID NO: 68 is the heavy chain variable region (V H ) of the OX40 agonist monoclonal antibody 11D4.

[0329] SEQ ID NO: 69 is the light chain variable region (V L ) of the OX40 agonist monoclonal antibody 11D4.

[0330] SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0331] SEQ ID NO: 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0332] SEQ ID NO: 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0333] SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0334] SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0335] Sequence number 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0336] Sequence number 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0337] Sequence number 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0338] Sequence number 78 is the heavy chain variable region (V H ) of the OX40 agonist monoclonal antibody 18D8.

[0339] Sequence number 79 is the light chain variable region (V L ) of the OX40 agonist monoclonal antibody 18D8.

[0340] Sequence number 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0341] Sequence number 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0342] Sequence number 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0343] Sequence number 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0344] Sequence number 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0345] Sequence number 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0346] Sequence number 86 is the heavy chain variable region (V H ) of the OX40 agonist monoclonal antibody Hu119-122.

[0347] Sequence number 87 is the variable region of the light chain (V L ) of the OX40 agonist monoclonal antibody Hu119-122.

[0348] Sequence number 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0349] Sequence number 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0350] Sequence number 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0351] Sequence number 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0352] Sequence number 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0353] Sequence number 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0354] Sequence number 94 is the variable region of the heavy chain (V H ) of the OX40 agonist monoclonal antibody Hu106-222.

[0355] Sequence number 95 is the variable region of the light chain (V L ) of the OX40 agonist monoclonal antibody Hu106-222.

[0356] Sequence number 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0357] Sequence number 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0358] Sequence number 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0359] Sequence number 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0360] Sequence number 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0361] Sequence number 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0362] Sequence number 102 is the OX40 ligand (OX40L) amino acid sequence.

[0363] Sequence number 103 is the soluble portion of the OX40L polypeptide.

[0364] Sequence number 104 is an alternative soluble portion of the OX40L polypeptide.

[0365] Sequence number 105 is the heavy chain variable region (V H ) of the OX40 agonist monoclonal antibody 008.

[0366] Sequence number 106 is the light chain variable region (V L ) of the OX40 agonist monoclonal antibody 008.

[0367] Sequence number 107 is the heavy chain variable region (V H ) of the OX40 agonist monoclonal antibody 011.

[0368] Sequence number 108 is the light chain variable region (V L ) of the OX40 agonist monoclonal antibody 011.

[0369] Sequence number 109 is the heavy chain variable region (V H ) of the OX40 agonist monoclonal antibody 021.

[0370] Sequence number 110 is the variable region of the light chain (V L ) of the OX40 agonist monoclonal antibody 021.

[0371] Sequence number 111 is the variable region of the heavy chain (V H ) of the OX40 agonist monoclonal antibody 023.

[0372] Sequence number 112 is the variable region of the light chain (V L ) of the OX40 agonist monoclonal antibody 023.

[0373] Sequence number 113 is the variable region of the heavy chain (V H ) of the OX40 agonist monoclonal antibody.

[0374] Sequence number 114 is the variable region of the light chain (V L ) of the OX40 agonist monoclonal antibody.

[0375] Sequence number 115 is the variable region of the heavy chain (V H ) of the OX40 agonist monoclonal antibody.

[0376] Sequence number 116 is the variable region of the light chain (V L ) of the OX40 agonist monoclonal antibody.

[0377] Sequence number 117 is the variable region of the heavy chain (V H ) of the humanized OX40 agonist monoclonal antibody.

[0378] Sequence number 118 is the variable region of the heavy chain (V H ) of the humanized OX40 agonist monoclonal antibody.

[0379] Sequence number 119 is the variable region of the light chain (V L ) of the humanized OX40 agonist monoclonal antibody.

[0380] SEQ ID NO: 120 is the variable light chain region (V L ) of a humanized OX40 agonist monoclonal antibody.

[0381] SEQ ID NO: 121 is the variable heavy chain region (V H ) of a humanized OX40 agonist monoclonal antibody.

[0382] SEQ ID NO: 122 is the variable heavy chain region (V H ) of a humanized OX40 agonist monoclonal antibody.

[0383] SEQ ID NO: 123 is the variable light chain region (V L ) of a humanized OX40 agonist monoclonal antibody.

[0384] SEQ ID NO: 124 is the variable light chain region (V L ) of a humanized OX40 agonist monoclonal antibody.

[0385] SEQ ID NO: 125 is the variable heavy chain region (V H ) of an OX40 agonist monoclonal antibody.

[0386] SEQ ID NO: 126 is the variable light chain region (V L ) of an OX40 agonist monoclonal antibody.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0387] I. Definitions 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 referred to herein are incorporated by reference in their entirety.

[0388] The term "in vivo" refers to events occurring within the body of a subject.

[0389] The term "in vitro" refers to events that occur outside the body of a subject. In vitro assays include cell-based assays in which live or dead cells are used, and may also include cell-free assays in which intact cells are not used.

[0390] The term "ex vivo" refers to events involving the performance of a treatment or procedure on cells, tissues, and / or organs removed from the body of a subject. Appropriately, the cells, tissues, and / or organs can be returned to the body of the subject by surgical or therapeutic means.

[0391] The term "rapid expansion" means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold), more preferably at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold), or most preferably at least about 100-fold over a period of one week. Some rapid expansion protocols are outlined below.

[0392] As used herein, "tumor-infiltrating lymphocytes" or "TIL" means a population of cells first obtained as white blood cells that have migrated from the bloodstream of a subject 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 TILs and secondary TILs. "Primary TIL" is that obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly obtained" or "freshly isolated"), and "secondary TIL" is any population of TIL cells that has been expanded or proliferated as discussed herein, including but not limited to bulk TIL and expanded TIL ("REP TIL" or "post-REP TIL"). TIL cell populations can include genetically modified TILs.

[0393] As used herein, "population of cells" (including TIL) refers to several cells sharing a common trait. Generally, the population is, in number, generally in the range of 1×10 6 to 1×10 10 , and different TIL populations contain different numbers. For example, the initial growth of primary TIL in the presence of IL-2 results in a bulk TIL population of approximately 1×10 8 cells. REP expansion is generally performed to provide a population of 1.5×10 9 to 1.5×10 10 cells for injection. In some embodiments, REP expansion is performed to provide a population of 2.3×10 10 to 13.7×10 10 .

[0394] As used herein, "cryopreserved TIL" means that any TIL of primary, bulk, or expanded (REP TIL) is processed and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere in this specification, including in the examples. For clarity, "cryopreserved TIL" can be distinguished from cryopreserved tissue samples that may be used as a source of primary TIL.

[0395] As used herein, "thawed cryopreserved TIL" means a population of TIL that was previously cryopreserved and then processed to return to a temperature above room temperature, including but not limited to cell culture temperature or a temperature at which the TIL can be administered to a patient.

[0396] TIL can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors to achieve treatment. TIL can generally be classified by the expression of one or more biomarkers among CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TIL can be defined functionally by its ability to infiltrate solid tumors upon reintroduction into a patient.

[0397] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such media can include media containing 7% - 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10". CS10 medium is a serum-free medium without animal components and contains DMSO.

[0398] The term "central memory T cell" refers to a subset of T cells that are CD45R0+ in humans and constitutively express CCR7 (CCR7 hi ) and CD62L (CD62 hi ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells mainly secrete IL-2 and CD40 L as effector molecules after TCR triggering. Central memory T cells are dominant in the CD4 compartment in the blood and are proportionally concentrated in lymph nodes and tonsils in humans.

[0399] The term "effector memory T cell" is CD45R0+ like central memory T cells, but has lost constitutive expression of CCR7 (CCR7 lo ), and has heterogeneous or low CD62L expression (CD62L lo) refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factor of central memory T cells includes BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines (including interferon γ, IL-4, and IL-5) after antigen stimulation. Effector memory T cells are dominant in the CD8 compartment in the blood and are proportionally concentrated in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry a large amount of perforin.

[0400] 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 method of the present invention. Closed systems include, but are not limited to, for example, sealed G containers. When tumor segments are added to a closed system, the system is not opened to the external environment until the TILs are ready to be administered to the patient.

[0401] The terms "fragmenting", "fragment", and "fragmented" as used herein to describe a process for destroying a tumor include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for destroying the physical structure of tumor tissue.

[0402] The term "fine needle aspiration" or FNA refers to a type of biopsy procedure that can be used in sampling or diagnostic procedures that involve sampling a tumor but not removing or excising it. In fine needle aspiration, for example, a 25- to 18-gauge hollow needle is inserted into the tumor or the area containing the tumor to obtain body fluids and cells (including tissues) for further analysis or expansion as described herein. In the case of FNA, cells are removed without maintaining the histological structure of the tissue cells. FNA may include TILs. In some cases, fine needle aspiration biopsy is performed using an ultrasound-guided fine needle aspiration biopsy needle. FNA needles are commercially available from Becton Dickinson, Covidien, etc.

[0403] The term "core biopsy" or "core needle biopsy" refers to a type of biopsy procedure that can be used for sampling or diagnostic procedures that involve tumor sampling but do not remove or excise the tumor. In core biopsy, for example, a 16- to 11-gauge hollow needle is inserted into the tumor or the area containing the tumor to obtain body fluids and cells (including tissues) for further analysis or expansion as described herein. In the case of core biopsy, due to the larger needle size compared to FNA, cells can be removed while maintaining to some extent the histological structure of the tissue cells. Core biopsy needles are generally of a gauge size that can maintain at least some of the histological structure of the tumor. Core biopsy may include TIL. In some cases, core needle biopsy is performed using a biopsy instrument, a vacuum-assisted core needle biopsy instrument, a core needle biopsy instrument under stereotactic guidance, a core needle biopsy instrument under ultrasound guidance, or a core needle biopsy instrument under MRI guidance, which are commercially available from Bard Medical, Becton Dickinson, etc.

[0404] The terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMC is a type of antigen-presenting cell), peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0405] The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBL is separated from whole blood or apheresis products from a donor. In some embodiments, PBL is separated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype such as the CD3+CD45+ T cell phenotype.

[0406] The term "anti-CD3 antibody" refers to an antibody or variant thereof, such as a monoclonal antibody, including human, humanized, chimeric, or murine antibodies 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, otrexup, teprotumumab, and besilesomab.

[0407] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies against the CD3 receptor in the T-cell antigen receptor of mature T cells, in a commercial form, such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 have been deposited with the American Type Culture Collection and assigned the ATCC accession number CRL8001. Hybridomas capable of producing OKT-3 have also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned the catalogue number 86022706.

Table 1

[0408] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial) Alternative IL-2 forms, as well as recombinant IL-2 forms commercially available from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b) and other commercial equivalents from other vendors are included. Aldesleukin (Des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form having 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 the pegylated IL-2 forms described herein, including the pegylated IL2 prodrug NKTR-214 available from Nektar Therapeutics (South San Francisco, CA, USA). NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, 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. Patents Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

Table 2

[0409] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. When activated by IL-4, Th2 T cells then produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgG 1 expression from B cells. Recombinant human IL-4 suitable for use in the present invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0410] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin 7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, which is a heterodimer consisting of the IL-7 receptor α and the common γ-chain receptor, and this is a series of signals important for T cell generation in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0411] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the 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 multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0412] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat.Rev.Drug.Disc.2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is , mainly natural killer T cells and activated human CD4 +It is produced by T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0413] When an "anti-tumor effective amount", "tumor inhibitory effective amount", or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in health status of the patient (subject). A pharmaceutical composition containing tumor-infiltrating lymphocytes described herein (e.g., secondary TIL or genetically modified cytotoxic lymphocytes) is 10 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 、10 5 ~10 10 、10 5 ~10 11 、10 6 ~10 10 、10 6 ~10 11 、10 7 ~10 11 、10 7 ~10 10 、10 8 ~10 11 、10 8 ~10 10 、10 9 ~10 11 、or 10 9 ~10 10It can be outlined that it can be administered at a dosage of [[number of cells]] cells / kg body weight (including all integer values within those ranges). Compositions of tumor-infiltrating lymphocytes (in some cases, including genetically modified cytotoxic lymphocytes) can also be administered multiple times at these dosages. Tumor-infiltrating lymphocytes (in some cases, including genetically modified cytotoxic lymphocytes) can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by a technician in the medical field by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0414] The terms "hematological malignancy", "hematopoietic malignancy" or terms of correlating meaning refer to cancers and tumors of mammalian hematopoietic and lymphoid tissues, including but not limited to blood, bone marrow, lymph nodes, and lymphoid tissues. Hematological malignancies are also referred to as "liquid tumors". Hematological malignancies can 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 lymphoma, and non-Hodgkin lymphoma. The term "B-cell hematological malignancy" refers to a hematological malignancy affecting B cells.

[0415] The term "solid tumor" usually refers to an abnormal mass of tissue that does not normally contain cysts or fluid areas. Solid tumors can be either 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 tissue structure of solid tumors includes interdependent tissue compartments containing parenchyma (cancer cells), and supporting stromal cells in which cancer cells are dispersed and which can provide a supportive microenvironment.

[0416] The term "liquid tumor" refers to an abnormal mass of cells that is inherently fluid. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0417] As used herein, the term "microenvironment" can refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. The tumor microenvironment as used herein refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from the host immune system, foster therapeutic resistance, and provide a niche for successful metastatic seeding," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that should be recognized by T cells, but due to immunosuppression by the microenvironment, tumor clearance by the immune system is rare.

[0418] In certain embodiments, the invention includes a method of treating cancer with a TIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TILs according to the invention. In some embodiments, a TIL population may be provided, and the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TILs according to the invention. In certain embodiments, the non-myeloablative chemotherapy is cyclophosphamide at 60 mg / kg / day for 2 days (27 and 26 days prior to TIL infusion), and fludarabine at 25 mg / m2 / day for 5 days (27 to 23 days prior to TIL infusion). In certain embodiments, after non-myeloablative chemotherapy and TIL infusion (day 0) according to the invention, the patient receives intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours up to physiological tolerance.

[0419] Experimental findings indicate that lymphocyte depletion prior to adoptive transfer of tumor - specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system (the "cytokine sink"). Accordingly, some embodiments of the present invention utilize a lymphocyte depletion step (sometimes referred to as "immunosuppressive conditioning") for the patient prior to introduction of the rTIL of the present invention.

[0420] As used herein, the terms "co - administration", "co - administering", "administered in combination with", "administered in combination with", "simultaneous", and "concurrent" encompass the administration of two or more pharmaceutical active ingredients (in a preferred embodiment of the present invention, for example, at least one potassium channel agonist in combination with multiple TILs) to a subject, such that both the pharmaceutical active ingredient and / or their metabolites are present in the subject simultaneously. Co - administration includes co - administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more pharmaceutical active ingredients are present. Co - administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0421] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein that is sufficient to achieve the intended use, including, but not limited to, treating a disease. The therapeutically effective amount can vary depending on the intended use (in vitro or in vivo), or the subject and condition being treated (e.g., the subject's weight, age, and gender), the severity of the condition, or the method of administration. This term also applies to a dosage that induces a specific response (e.g., reduction of platelet adhesion and / or cell migration) in target cells. The specific dosage will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is carried.

[0422] Terms such as "treatment", "treating", "treat" refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or side effects caused by the disease. As used herein "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes (a) preventing a disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease, (b) suppressing the disease, i.e., arresting its development or progression, and (c) alleviating the disease, i.e., causing regression of the disease and / or alleviating one or more symptoms of the disease. "treatment" is also intended to include the delivery of an agent for providing a pharmacological effect even in the absence of a disease or condition. For example, "treatment" includes the delivery of a composition that can induce an immune response or confer immunity, such as in the case of a vaccine, in the absence of a medical condition.

[0423] The term "heterologous" when used in reference to a portion of a nucleic acid or protein indicates that the nucleic acid or protein contains two or more sub-sequences that are not found in nature in the same relationship to each other. For example, a nucleic acid is typically recombinantly produced and arranged to create a novel functional nucleic acid, such as a promoter from one source and a coding region from another source, or two or more sequences from unrelated genes arranged to create a coding region from different sources. Similarly, a heterologous protein indicates that the protein contains two or more sub-sequences that are not found in nature in the same relationship to each other (e.g., a fusion protein).

[0424] The terms "sequence identity," "percent identity," and "percent sequence identity" (or synonyms thereof, such as "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence without considering any conservative amino acid substitutions as part of sequence identity (introducing gaps if necessary). The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art that can be used to obtain an alignment of amino acid or nucleotide sequences. Programs suitable for determining percent sequence identity include, for example, the BLAST programs available from the BLAST website of the National Center for Biotechnology Information of the U.S. government. The comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR are additional publicly available software programs that can be used to align sequences. One of ordinary skill in the art can determine the appropriate parameters for maximum alignment with a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0425] As used herein, the term "variant" includes, but is not limited to, a protein, antibody, or fusion protein that contains an amino acid sequence that differs from the amino acid sequence of a reference antibody, protein, or fusion protein only by one or more substitutions, deletions, and / or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody, protein, or fusion protein. A variant may include 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, the substitution of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody, protein, or fusion protein to specifically bind to the antigen. The term variant also includes pegylated antibodies or proteins.

[0426] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to lymphocytes that leave the bloodstream of a subject and infiltrate into the tumor. TILs are a group of cells that are initially obtained as leukocytes that migrate into tumors. + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly obtained" or "freshly isolated"), and "secondary TILs" are any TIL cell populations expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, a second expanded TIL or a second additional expanded TIL (e.g., as described in step D of FIG. 1, including TILs referred to as reREP TILs).

[0427] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors to achieve therapy. TILs can generally be classified by the expression of one or more biomarkers among CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into the patient. TILs can be further characterized by potency; for example, if interferon (IFN) release exceeds about 50 pg / mL, about 100 pg / mL, about 150 pg / mL, or about 200 pg / mL, the TILs can be considered potent. For example, if interferon (IFNγ) release exceeds about 50 pg / mL, about 100 pg / mL, about 150 pg / mL, or about 200 pg / mL, about 300 pg / mL, about 400 pg / mL, about 500 pg / mL, about 600 pg / mL, about 700 pg / mL, about 800 pg / mL, about 900 pg / mL, about 1000 pg / mL, the TILs can be considered potent.

[0428] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for pharmaceutical active ingredients is well known in the art. Their use in the therapeutic compositions of the present invention is contemplated, except where any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the pharmaceutical active ingredient. Additional pharmaceutical active ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0429] The terms "about" and "approximately" mean within a statistically significant range of a value. Such range can be within one digit of a given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The tolerance included by the term "about" or "approximately" depends on the particular system under study and can be readily understood by a person skilled in the art. Further, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not exact and need not be exact, but may be approximate and / or larger or smaller than indicated, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to a person skilled in the art. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are "about" or "approximately", whether or not so explicitly stated. Note that arrangements may be employed that adopt embodiments of very different sizes, shapes, and dimensions.

[0430] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the scope of the claims with respect to whether additional claim elements or steps not recited, in their original form and in any amended form, if present, are excluded from the scope of the claims. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, method, steps, or materials. The term "consisting of" excludes any element, step, or material not specified in the claims, and in the latter case, also excludes impurities ordinarily associated with the specified materials. The term "consisting essentially of" limits the scope of the claims to the specified element, step, or materials, and those that do not materially affect the basic and novel characteristics of the claimed invention. All compositions, methods, and kits described herein embodying the invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0431] II. TIL manufacturing process (embodiment of the GEN3 process, optionally including synthetic medium) Without being limited to any particular theory, it is believed that the method of the present invention enables the preparation of expanded T cells that retain a "younger" phenotype through a first priming expansion that primes the activation of T cells, followed by a second rapid expansion that promotes the activation of T cells. Thus, it is expected that the expanded T cells of the present invention will exhibit higher cytotoxicity against cancer cells than T cells expanded by other methods. In particular, T cell activation primed by an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally exposure to antigen-presenting cells (APCs) as taught by the method of the present invention, and subsequently promoted by additional anti-CD-3 antibody (e.g., OKT-3), IL-2, and subsequent exposure to APCs, limits or avoids the maturation of T cells in culture and produces a T cell population with a less mature phenotype, and these T cells are thought to be less exhausted by expansion in culture and exhibit higher cytotoxicity against cancer cells. In some embodiments, the second rapid expansion step is divided into multiple steps, (a) performing the second rapid expansion by culturing T cells for about 3 to 4 days in a first vessel, e.g., a small-scale culture in a G-REX 100MCS vessel, and then (b) transferring the T cells from the small-scale culture to a second vessel larger than the first vessel, e.g., a G-REX 500MCS vessel, and culturing the T cells from the small-scale culture for about 4 to 7 days in a larger-scale culture in the second vessel to achieve scale-up of the culture. In some embodiments, the rapid expansion step is divided into multiple steps, (a) performing the second rapid expansion by culturing T cells for 3 to 4 days in a first vessel, e.g., a first small-scale culture in a G-REX 100MCS vessel, and then (b) achieving scale-out of the culture by transferring and distributing the T cells from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of the same size as the first vessel, and in each second vessel, a portion of the T cells from the first small-scale culture transferred to such second vessel is cultured for about 4 to 7 days in a second small-scale culture.In some embodiments, the rapid expansion step is divided into multiple steps, and (a) the second rapid expansion is performed by culturing T cells in a first container, e.g., a small-scale culture in a G-REX 100MCS container, for about 3 to 4 days, and then (b) the T cells derived from the small-scale culture are transferred and distributed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are larger in size than the first container, e.g., G-REX 500MCS containers, to achieve scale-out and scale-up of the culture. In each second container, a portion of the T cells derived from the small-scale culture transferred to such second container is cultured in a larger-scale culture for about 4 to 7 days. In some embodiments, the rapid expansion step is divided into multiple steps, and (a) the T cells are cultured in a first container, e.g., a small-scale culture in a G-REX 100MCS container for about 4 days to perform the second rapid expansion, and then (b) the T cells derived from the small-scale culture are transferred and distributed to 2, 3, or 4 second containers that are larger in size than the first container, e.g., G-REX 500MCS containers, to achieve scale-out and scale-up of the culture. In each second container, a portion of the T cells derived from the small-scale culture transferred to such second container is cultured in a larger-scale culture for about 5 days. The second rapid expansion is performed by culturing T cells in a first container, e.g., a small-scale culture in a G-REX 100MCS container for about 4 days, and then (b) the T cells derived from the small-scale culture are transferred and distributed to 2, 3, or 4 second containers that are larger in size than the first container, e.g., G-REX 500MCS containers, to achieve scale-out and scale-up of the culture. In each second container, a portion of the T cells derived from the small-scale culture transferred to such second container is cultured in a larger-scale culture for about 5 days.

[0432] In some embodiments, the second rapid expansion is performed after the activation of the T cells brought about by the first priming expansion begins to decrease, abate, decline, or become quiescent.

[0433] In some embodiments, the second rapid expansion is performed after the activation of T cells brought about by the first priming expansion has decreased by exactly or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0434] In some embodiments, the second rapid expansion is performed after the activation of T cells brought about by the first priming expansion has decreased by a percentage in the range of exactly or approximately 1% to 100%.

[0435] In some embodiments, the second rapid expansion is performed after the activation of T cells brought about by the first priming expansion has decreased by a percentage in the range of exactly or approximately 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100%.

[0436] In some embodiments, the second rapid expansion is performed after the activation of T cells brought about by the first priming expansion has decreased by at least exactly or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0437] In some embodiments, the second rapid expansion is performed after the activation of T cells brought about by the first priming expansion has decreased by at most exactly or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% and then.

[0438] In some embodiments, the decrease in the activation of T cells brought about by the first priming expansion is determined by a decrease in the amount of interferon-γ released by the T cells in response to antigenic stimulation.

[0439] In some embodiments, the first priming expansion of T cells occurs over a period of up to exactly or about 7 or about 8 days.

[0440] In some embodiments, the first priming expansion of T cells occurs over a period of up to exactly or about 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0441] In some embodiments, the first priming expansion of T cells occurs over a period of 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0442] In some embodiments, the second rapid expansion of T cells occurs over a period of up to exactly or about 11 days.

[0443] In some embodiments, the second rapid expansion of T cells occurs over a period of up to exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0444] In some embodiments, the second rapid expansion of T cells occurs over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0445] In some embodiments, the first priming expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 7 days, and the second rapid expansion of T cells occurs over a period of exactly or about 1 day to exactly or about 11 days.

[0446] In some embodiments, the first priming expansion of T cells occurs over a period of up to exactly or about 1, 2, 3, 4, 5, 6, 7, or 8 days, and the second rapid expansion of T cells occurs over a period of up to exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days.

[0447] In some embodiments, the first priming expansion of T cells is carried out during a period of exactly or about 1 day to exactly or about 8 days, and the second rapid expansion of T cells is carried out during a period of exactly or about 1 day to exactly or about 9 days.

[0448] In some embodiments, the first priming expansion of T cells is carried out during an 8-day period, and the second rapid expansion of T cells is carried out during a 9-day period.

[0449] In some embodiments, the first priming expansion of T cells is carried out during a period of exactly or about 1 day to exactly or about 7 days, and the second rapid expansion of T cells is carried out during a period of exactly or about 1 day to exactly or about 9 days.

[0450] In some embodiments, the first priming expansion of T cells is carried out during a 7-day period, and the second rapid expansion of T cells is carried out during a 9-day period.

[0451] In some embodiments, the T cells are tumor-infiltrating lymphocytes (TILs).

[0452] In some embodiments, the T cells are marrow-infiltrating lymphocytes (MILs).

[0453] In some embodiments, the T cells are peripheral blood lymphocytes (PBLs).

[0454] In some embodiments, the T cells are obtained from a donor suffering from cancer.

[0455] In some embodiments, the T cells are TILs obtained from a tumor excised from a patient suffering from cancer.

[0456] In some embodiments, the T cells are MILs obtained from the bone marrow of a patient suffering from a hematological malignancy.

[0457] In some embodiments, the T cells are PBLs obtained from donor-derived peripheral blood mononuclear cells (PBMCs). In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematological malignancy.

[0458] In certain aspects of the present disclosure, immune effector cells, such as T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as Ficoll separation. In a preferred aspect, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically include lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis are washed to remove the plasma fraction and optionally placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution may lack calcium, lack magnesium, or lack many, but not all, divalent cations. In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL gradient or by counterflow centrifugal elution.

[0459] In some embodiments, the T cells are PBLs isolated from whole blood or apheresis products enriched with donor-derived lymphocytes. In some embodiments, the donor has cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor is. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has a hematological malignancy. In some embodiments, the PBLs are isolated from whole blood or apheresis products enriched with lymphocytes by using positive or negative selection methods, i.e., removing PBLs using a marker(s), e.g., CD3+CD45+, for the T cell phenotype, or removing non-T cell phenotype cells to leave the PBLs. In other embodiments, the PBLs are isolated by gradient centrifugation. At the time of isolating PBLs from the donor tissue, the first priming expansion of the PBLs can be initiated by seeding a suitable number of isolated PBLs (in some embodiments, approximately 1×10 7 PBLs) under the first priming expansion culture according to any of the first priming expansion steps of the methods described herein.

[0460] An exemplary TIL process known as Process 3 (also referred to herein as GEN3), which includes some of these features, is shown in FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), and some of the advantages of this embodiment of the invention over Process 2A are described in FIGS. 1, 2, 30, and 31 (in particular, for example, FIGS. 1B and / or 1C). Two embodiments of Process 3 are shown in FIGS. 1 and 30 (in particular, for example, FIGS. 1B and / or 1C). Process 2A or Gen2 is also described in U.S. Patent Publication No. 2018 / 0280436, which is hereby incorporated by reference in its entirety.

[0461] As discussed and generally outlined herein, TILs are harvested from a patient sample and engineered to expand their numbers prior to transplantation into the patient using a TIL expansion process described herein and referred to as Gen3. In some embodiments, the TILs can optionally be genetically engineered as discussed below. In some embodiments, the TILs can be cryopreserved before or after expansion. Upon thawing, they can be restimulated to enhance metabolism prior to injection into the patient.

[0462] In some embodiments, as discussed in detail below and in the Examples and Figures, the first priming expansion (including the process referred to herein as Pre-Rapid Expansion (Pre-REP) and shown as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-8 days, and the second rapid expansion (including the process referred to herein as Rapid Expansion Protocol (REP) and shown as step D in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-9 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the first priming expansion (including a process referred to herein as Pre-Rapid Expansion (Pre-REP) and shown as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-8 days, and the second rapid expansion (including a process referred to herein as Rapid Expansion Protocol (REP) and shown as step D in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-8 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the first priming extension (including the process referred to herein as pre-rapid expansion (pre-REP) and the process shown as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-7 days, and the second rapid expansion (including the process referred to herein as rapid expansion protocol (REP) and the process shown as step D in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-9 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the first priming extension (including the process referred to herein as pre-rapid expansion (pre-REP) and the process shown as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-7 days, and the second rapid expansion (including the process referred to herein as rapid expansion protocol (REP) and the process shown as step D in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1-9 days. The process shown as step D) is from 1 to 10 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is shortened to 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is from 7 to 9 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is from 8 to 9 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is shortened to 7 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is from 7 to 8 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is shortened to 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is 8 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is 9 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is 8 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1, particularly, e.g., FIGS. 1B and / or 1C) is 10 days.In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 7 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 7 to 10 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 7 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 8 to 10 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 7 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 9 to 10 days. In some embodiments, the first priming expansion (e.g., the expansion described as step B in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is shortened to 7 days, and the second rapid expansion (e.g., the expansion described as step D in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 7 to 9 days. In some embodiments, as will be discussed in detail below as well as in the examples and figures, the combination of the first priming expansion and the second rapid expansion (e.g., the expansion described as step B and the expansion described as step D in FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C)) is 14 to 16 days. In particular, certain embodiments of the present invention are contemplated to include a first priming expansion step in which TILs are activated by exposure to an anti-CD3 antibody, e.g., OKT-3, in the presence of IL-2, or exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody, e.g., OKT-3. In certain embodiments, the TILs activated in the first priming expansion step as described above are a first TIL population, i.e., a primary cell population.

[0463] The following "Steps" notations A, B, C, etc. refer to non-limiting examples in FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), and to certain non-limiting embodiments described herein. The order of steps in the following and in FIG. 1 (in particular, for example, FIGS. 1B and / or 1C) is exemplary, and any combination or order of steps, as well as additional steps, repetitions of steps, and / or omissions of steps are contemplated by the methods disclosed in this application and in this specification. and additional steps, repetitions of steps, and / or omissions of steps are contemplated by the methods disclosed in this application and in this specification.

[0464] A. Step A: Obtain a tumor sample from a patient Generally, TILs are obtained initially from a patient's tumor sample ("primary TIL") or from circulating lymphocytes such as peripheral blood lymphocytes containing peripheral blood lymphocytes with TIL-like characteristics, and then, as described herein, are expanded to a larger population for further manipulation, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

[0465] A patient's tumor sample can generally be obtained using methods known in the art by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. Generally, the tumor sample can be derived from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be a liquid tumor such as a tumor obtained from a hematological malignancy. Solid tumors can include, but are not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). 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 multiforme (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, useful TILs are obtained from malignant melanoma tumors because malignant melanoma tumors have been reported to have particularly high levels of TILs.

[0466] Once obtained, the tumor sample is generally fragmented into pieces of 1 to about 8 mm using sharp dissection 3 and pieces of about 2 to 3 mm 3 are particularly useful. TILs are cultured from these fragments using enzymatic tumor digest. Such tumor digest can be produced by incubation in an enzymatic 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 dissociator). The tumor digest is produced by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, then incubating at 37° C. in 5% CO 2 for 30 minutes, and subsequently repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only small tissue pieces remain. At the end of this process, if a large number of red blood cells or dead cells are included in the cell suspension, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference, can be used. Any of the aforementioned methods can be used in any of the embodiments described herein for a method of expanding TILs or a method of treating cancer.

[0467] As shown above, in some embodiments, TILs are derived from solid tumors. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as an entire tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours at 37 °C, 5% CO 2 and is digested. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours at 37 °C, 5% CO 2 and is digested with rotation. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37 °C, 5% CO 2 with constant rotation. In some embodiments, the entire tumor is combined with the enzyme to form a tumor digestion reaction mixture.

[0468] In some embodiments, the tumor is reconstituted with lyophilized enzyme in sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0469] In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 10-fold working stock of 100 mg / mL.

[0470] In some embodiments, the enzyme mixture contains DNAse. In some embodiments, the working stock of DNAse is a 10-fold working stock of 10,000 IU / mL.

[0471] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10-fold working stock of 10 mg / mL.

[0472] In some embodiments, the enzyme mixture comprises 10 mg / mL of collagenase, 1000 IU / mL of DNAse, and 1 mg / mL of hyaluronidase.

[0473] In some embodiments, the enzyme mixture comprises 10 mg / mL of collagenase, 500 IU / mL of DNAse, and 1 mg / mL of hyaluronidase.

[0474] Generally, a cell suspension obtained from a tumor is referred to as a "primary cell population" or "freshly obtained" or "freshly isolated" cell population. In certain embodiments, the freshly obtained TIL cell population is exposed to a cell culture medium containing antigen-presenting cells, IL-12, and OKT-3.

[0475] In some embodiments, fragmentation includes physical fragmentation, such as 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, TIL can be cultured initially from enzyme tumor digest and tumor fragments obtained from a patient. In one embodiment, TIL can be cultured initially from enzyme tumor digest and tumor fragments obtained from a patient.

[0476] In some embodiments where the tumor is a solid tumor, after the tumor sample is obtained, for example, as provided in step A (Figure 1 (particularly, for example, Figure 1B and / or Figure 1C)), the tumor undergoes physical fragmentation. In some embodiments, the fragmentation occurs prior to cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, or more fragments or pieces are placed in each container for the first priming expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first priming expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first priming expansion. In some embodiments, the plurality of fragments includes from about 4 to about 50 fragments, and each fragment has a volume of about 27 mm 3 In some embodiments, the plurality of fragments includes from about 30 to about 60 fragments, and the total volume is from about 1300 mm 3 to about 1500 mm 3 In some embodiments, the plurality of fragments includes about 50 fragments, and the total volume is about 1350 mm 3 In some embodiments, the plurality of fragments includes about 50 fragments, and the total mass is from about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments includes about 4 fragments.

[0477] In some embodiments, TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are from about 1 mm 3 to 10 mm 3 In some embodiments, the tumor fragments are from about 1 mm 3 to 8 mm 3 In some embodiments, the tumor fragments are from about 1 mm 3 In some embodiments, the tumor fragments are about 2 mm3 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 fragment is 1 - 4 mm × 1 - 4 mm × 1 - 4 mm. In some embodiments, the tumor fragment is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2 mm × 2 mm × 2 mm. In some embodiments, the tumor fragment is 3 mm × 3 mm × 3 mm. In some embodiments, the tumor fragment is 4 mm × 4 mm × 4 mm.

[0478] In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic, and / or fatty tissue on each small piece. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue on each small piece. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each small piece. In some embodiments, the tumor is fragmented to minimize the amount of fatty tissue on each small piece. In certain embodiments, the tumor fragmentation step is an in vitro or ex vivo method.

[0479] In some embodiments, fragmentation of the tumor is performed to maintain the internal structure of the tumor. In some embodiments, fragmentation of the tumor is performed without performing a scraping operation with a scalpel. In some embodiments, TILs are obtained from tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor can be mechanically dissociated for approximately 1 minute. Thereafter, the solution is incubated at 37°C in 5% CO 2 for 30 minutes, and then can be mechanically disrupted again for approximately 1 minute. After incubation at 37°C in 5% CO 2 for 30 minutes again, the tumor can be subjected to a third mechanical disruption for approximately 1 minute. In some embodiments, if large tissue pieces are present, one or two additional mechanical dissociations are applied to the sample, regardless of whether it is incubated at 37°C in 5% CO 2 for an additional 30 minutes after the third mechanical disruption. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0480] In some embodiments, the cell suspension before the first priming expansion step is referred to as a "primary cell population" or "newly obtained" or "newly isolated" cell population.

[0481] In some embodiments, the cells can be optionally frozen after sample isolation (e.g., after obtaining a tumor sample and / or after obtaining a cell suspension from the tumor sample), and cryopreserved before entering the expansion described in step B, which is described in more detail below and illustrated also in FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C).

[0482] 1. Core / Biopsy-Derived TILs In some embodiments, TILs are first obtained from a patient tumor sample (“primary TILs”) obtained by a core biopsy or similar procedure and then expanded to a larger population for further manipulation, cryopreserved optionally, and optionally evaluated for phenotypic and metabolic parameters as described herein.

[0483] In some embodiments, a patient tumor sample can generally be obtained using methods known in the art by a core biopsy, a fine needle biopsy, a needle biopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells. Generally, the tumor sample can be derived from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample can also be a liquid tumor, such as a tumor obtained from a hematological malignancy. In some embodiments, the sample can be derived from multiple small tumor samples or biopsies. In some embodiments, the sample can include multiple tumor samples from a single tumor from the same patient. In some embodiments, the sample can include multiple tumor samples from 1, 2, 3, or 4 tumors from the same patient. In some embodiments, the sample can include multiple tumor samples from multiple tumors from the same patient. Solid tumors can be any type of cancer, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (e.g., including head and neck squamous cell carcinoma (HNSCC)), glioblastoma multiforme (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer (NSCLC). In some embodiments, useful TILs are obtained from malignant melanoma tumors because malignant melanoma tumors have been reported to have particularly high levels of TILs.

[0484] Generally, a cell suspension obtained from a tumor core or fragment is referred to as a "primary cell population" or a "newly obtained" or "newly isolated" cell population. In certain embodiments, the newly obtained TIL cell population is exposed to a cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

[0485] In some embodiments, if the tumor is metastatic and the primary lesion has been efficiently treated / removed in the past, removal of one of the metastatic lesions may be required. In some embodiments, the least invasive approach is to remove a skin lesion or, if available, a lymph node in the neck or axillary region. In some embodiments, the skin lesion is removed or its biopsy is removed. In some embodiments, the lymph node or its biopsy is removed. In some embodiments, a lung or liver metastatic lesion, or an intraperitoneal or thoracic lymph node or its biopsy may be used.

[0486] In some embodiments, the tumor is a melanoma. In some embodiments, the melanoma biopsy includes a mole or a part thereof.

[0487] In some embodiments, the biopsy is a punch biopsy. In some embodiments, the punch biopsy is obtained with a circular blade pushed into the skin. In some embodiments, the punch biopsy is obtained with a circular blade pushed into the skin around a suspicious mole. In some embodiments, the punch biopsy is obtained with a circular blade pushed into the skin, and a round piece of skin is collected. In some embodiments, the biopsy is a punch biopsy, and a round portion of the tumor is collected.

[0488] In some embodiments, the biopsy is an excisional biopsy. In some embodiments, the biopsy is an excisional biopsy, and the mole or the entire growth is removed. In some embodiments, the biopsy is an excisional biopsy, and the mole or the entire growth is removed together with a small margin of apparently normal skin.

[0489] In some embodiments, the biopsy is an incisional biopsy. In some embodiments, the biopsy is an incisional biopsy and only the mole or the most irregular part of the growth is taken. In some embodiments, the biopsy is an incisional biopsy and the incisional biopsy is used when other techniques cannot be achieved, such as when the suspicious mole is very large.

[0490] In some embodiments, the biopsy is a lung biopsy. In some embodiments, the biopsy is obtained by bronchoscopy. Generally, in bronchoscopy, the patient is anesthetized, a small instrument is passed through the nose or mouth, down the throat into the bronchial passage, and a small amount of tissue is taken using the small instrument there. In some embodiments where the tumor or growth cannot be reached by bronchoscopy, a transthoracic needle biopsy may be used. Generally, in the case of a transthoracic needle biopsy as well, the patient is anesthetized and the needle is inserted directly through the skin into the suspected location to take a small tissue sample. In some embodiments, a transthoracic needle biopsy may require interventional radiology (e.g., the use of X-ray or CT scans to guide the needle). In some embodiments, the biopsy is obtained by a needle biopsy. In some embodiments, the biopsy is obtained by endoscopic ultrasound (e.g., an endoscope with illumination, placed in the esophagus through the mouth). In some embodiments, the biopsy is obtained surgically.

[0491] In some embodiments, the biopsy is a head and neck biopsy. In some embodiments, the biopsy is an incisional biopsy. In some embodiments, the biopsy is an incisional biopsy and a small tissue piece is excised from an area where it appears abnormal. In some embodiments, if the abnormal area is easily accessible, the sample can be taken without hospitalization. In some embodiments, if the tumor is deeper in the mouth or throat, the biopsy may need to be performed in an operating room using general anesthesia. In some embodiments, the biopsy is an excisional biopsy. In some embodiments, the biopsy is an excisional biopsy in which the entire area is removed. In some embodiments, the biopsy is a fine needle aspiration (FNA). In some embodiments, the biopsy is a fine needle aspiration (FNA) and a very thin needle attached to a syringe is used to extract (aspirate) cells from the tumor or mass. In some embodiments, the biopsy is a punch biopsy. In some embodiments, the biopsy is a punch biopsy and a punch forceps is used to take a piece of the suspicious area.

[0492] In some embodiments, the biopsy is a cervical biopsy. In some embodiments, the biopsy is obtained by colposcopy. Generally, colposcopy involves the use of an illuminated magnifying instrument (colposcope) attached to a binocular magnifier, and then this is used to biopsy a small section of the surface of the cervix. In some embodiments, the biopsy is a conization / cone biopsy. In some embodiments, the biopsy is a conization / cone biopsy and an outpatient procedure may be required to obtain a larger tissue piece from the cervix. In some embodiments, in addition to helping to confirm the diagnosis, the cone biopsy can be the initial treatment.

[0493] The term "solid tumor" generally refers to an abnormal mass of tissue that does not typically include cysts or liquid areas. Solid tumors can be either 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, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The tissue structure of a solid tumor includes interdependent tissue compartments that contain parenchyma (cancer cells), and supportive stromal cells in which the cancer cells are dispersed and that can provide a supportive microenvironment.

[0494] In some embodiments, tumor-derived samples are obtained as fine needle aspirates (FNAs), core biopsies, or small biopsies (including, e.g., punch biopsies). In some embodiments, the sample is first placed into a G-Rex 10. In some embodiments, if one or two core biopsies and / or small biopsy samples are present, the sample is first placed into a G-Rex 10. In some embodiments, if three, four, five, six, eight, nine, or ten or more core biopsies and / or small biopsy samples are present, the sample is first placed into a G-Rex 100. In some embodiments, if three, four, five, six, eight, nine, or ten or more core biopsies and / or small biopsy samples are present, the sample is first placed into a G-Rex 500.

[0495] An FNA can be obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervix, ovary, pancreas, glioblastoma, colorectal, and sarcoma. In some embodiments, the FNA is obtained from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the patient with NSCLC has previously undergone surgical treatment.

[0496] The TILs described herein can be obtained from FNA samples. In some cases, the FNA samples are obtained or isolated from a patient using a thin gauge needle in the range of 18-gauge to 25-gauge needles. The thin gauge needle can be 18-gauge, 19-gauge, 20-gauge, 21-gauge, 22-gauge, 23-gauge, 24-gauge, or 25-gauge. In some embodiments, the patient-derived FNA sample can contain at least 400,000 TILs, for example, 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

[0497] In some cases, the TILs described herein are obtained from core biopsy samples. In some cases, the core biopsy samples are obtained or isolated from a patient using a surgical or medical needle in the range of 11-gauge to 16-gauge needles. The needle can be 11-gauge, 12-gauge, 13-gauge, 14-gauge, 15-gauge, or 16-gauge. In some embodiments, the patient-derived core biopsy sample can contain at least 400,000 TILs, for example, 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

[0498] Generally, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

[0499] In some embodiments, the TILs are not obtained from tumor digests. In some embodiments, the solid tumor core is not fragmented.

[0500] In some embodiments, TILs are obtained from tumor digests. In some embodiments, the tumor digest is generated by incubation in an enzyme medium such as, but not limited to, RPMI 1640 (2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor can be mechanically dissociated for approximately 1 minute. The solution is then incubated at 37 °C in 5% CO 2 for 30 minutes, after which it can be mechanically disrupted again for approximately 1 minute. After incubating again at 37 °C in 5% CO 2 for 30 minutes, the tumor can be subjected to a third mechanical disruption for approximately 1 minute. In some embodiments, if large tissue pieces are present, one or two additional mechanical dissociations are applied to the sample, regardless of whether it is incubated at 37 °C in 5% CO 2 for an additional 30 minutes after the third mechanical disruption. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed at the end of the final incubation to remove these cells.

[0501] 2. Method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood PBL Method 1. In certain embodiments of the invention, PBLs are expanded using the processes described herein. In certain embodiments of the invention, the method includes obtaining a PBMC sample from whole blood. In certain embodiments, the method includes enriching T cells by isolating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. In certain embodiments, the method includes enriching T cells by isolating pure T cells from PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

[0502] In certain embodiments of the present invention, PBL method 1 is performed as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. T cells are isolated using a human Pan T cell isolation kit and an LS column (Miltenyi Biotec).

[0503] PBL method 2. In certain embodiments of the present invention, PBL is expanded using PBL method 2, which includes obtaining a PBMC sample from whole blood. T cells derived from PBMCs are enriched by incubating the PBMCs at 37°C for at least 3 hours and then isolating the non-adherent cells.

[0504] In certain embodiments of the present invention, PBL method 2 is performed as follows: On day 0, the cryopreserved PMBC sample is thawed, and the PBMC cells are seeded at 6 million cells / well in a 6-well plate in CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, the non-adherent cells, which are PBL, are removed and counted.

[0505] PBL method 3. In certain embodiments of the present invention, PBL is expanded using PBL method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are isolated using CD19+ selection, and T cells are selected using negative selection of the non-CD19+ fraction of the PBMC sample.

[0506] In certain embodiments of the present invention, PBL method 3 is performed as follows: On day 0, the cryopreserved PBMCs obtained from peripheral blood are thawed and counted. CD19+ B cells are sorted using a CD19 Multisort kit, human (Miltenyi Biotec). Among the non-CD19+ cell fraction, T cells are purified using a human Pan T cell isolation kit and an LS column (Miltenyi Biotec).

[0507] In certain embodiments, PBMCs are isolated from whole blood samples. In certain embodiments, the PBMC sample is used as a starting material for expanding PBLs. In certain embodiments, the sample is cryopreserved prior to the expansion process. In another embodiment, a fresh sample is used as a starting material for expanding PBLs. In certain embodiments of the present invention, T cells are isolated from PBMCs using methods known in the art. In certain embodiments, the T cells are isolated using a human Pan T cell isolation kit and an LS column. In certain embodiments of the present invention, T cells are isolated from PBMCs using antibody selection methods known in the art, such as negative selection of CD19.

[0508] In certain embodiments of the present invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify non-adherent cells. In certain embodiments of the present invention, the incubation time is about 3 hours. In certain embodiments of the present invention, the temperature is about 37°C. Thereafter, the non-adherent cells are expanded using the above process.

[0509] In some embodiments, the PBMC sample is from a subject or patient optionally pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and has been treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more. In another embodiment, the PBMC is from a patient currently receiving an ITK inhibitor regimen such as ibrutinib.

[0510] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and is refractory to treatment with a kinase inhibitor or an ITK inhibitor such as ibrutinib.

[0511] In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but is no longer receiving treatment with a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but has not received treatment with a kinase inhibitor or an ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year, or longer. In another embodiment, the PBMC is from a patient who has been previously exposed to an ITK inhibitor but has not been treated within at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0512] In certain embodiments of the invention, on day 0, cells are selected for and sorted according to CD19+. In certain embodiments of the invention, the selection is performed using antibody-conjugated beads. In certain embodiments of the invention, pure T cells are isolated from PBMC on day 0.

[0513] In certain embodiments of the invention, for patients not pre-treated with ibrutinib or other ITK inhibitors, a 10 - 15 mL buffy coat yields approximately 5×10 9 PBMC, which in turn yields approximately 5.5×10 7 PBL.

[0514] In certain embodiments of the invention, for patients pre-treated with ibrutinib or other ITK inhibitors, the expansion process yields approximately 20×10 9 PBL. In certain embodiments of the invention, 40.3×10 6Individual PBMCs produce approximately 4.7×10 5 PBLs.

[0515] In any of the foregoing embodiments, PBMCs can be obtained from whole blood samples, by apheresis, from buffy coats, or by any other method known in the art for obtaining PBMCs.

[0516] 3. Method for expanding myeloid infiltrating lymphocytes (MIL) from bone marrow-derived PBMCs MIL method 3. In certain embodiments of the present invention, this method involves obtaining PBMCs from bone marrow. On day 0, PBMCs are selected for CD3+ / CD33+ / CD20+ / CD14+, sorted, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is returned and added to the selected cell fraction.

[0517] In certain embodiments of the present invention, MIL method 3 is performed as follows: On day 0, a cryopreserved PBMC sample is thawed and the PBMCs are counted. The cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using a selected S3e cell (Bio-Rad). The cells are sorted into two fractions: an immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and an AML blast fraction (non-CD3+CD33+CD20+CD14+).

[0518] In certain embodiments of the present invention, PBMCs are obtained from bone marrow. In certain embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, needle biopsy, or other similar means known in the art. In certain embodiments, PBMCs are fresh. In another embodiment, PBMCs are cryopreserved.

[0519] In certain embodiments of the present invention, the MIL is expanded from 10 to 50 mL of bone marrow aspirate. In certain embodiments of the present invention, 10 mL of bone marrow aspirate is obtained from a patient. In another embodiment, 20 mL of bone marrow aspirate is obtained from a patient. In another embodiment, 30 mL of bone marrow aspirate is obtained from a patient. In another embodiment, 40 mL of bone marrow aspirate is obtained from a patient. In another embodiment, 50 mL of bone marrow aspirate is obtained from a patient.

[0520] In certain embodiments of the present invention, the number of PBMCs produced from about 10 to 50 mL of bone marrow aspirate is about 5×10 7 ~ about 10×10 7 PBMCs. In another embodiment, the number of PMBCs produced is about 7×10 7 PBMCs.

[0521] In certain embodiments of the present invention, about 5×10 7 ~ about 10×10 7 PBMCs produce about 0.5×10 6 ~ about 1.5×10 6 MILs. In certain embodiments of the present invention, about 1×10 6 MILs are produced.

[0522] In certain embodiments of the present invention, 12×10 6 PBMCs obtained from bone marrow aspirate produce approximately 1.4×10 5 MILs.

[0523] In any of the foregoing embodiments, the PBMCs can be obtained from a whole blood sample, from bone marrow, by apheresis, from a buffy coat, or by any other method known in the art for obtaining PBMCs.

[0524] B. Step B: First priming expansion In some embodiments, the method provides younger TILs, which may provide additional therapeutic benefit over older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to the subject / patient). Characteristics of younger TILs are described in the literature, e.g., 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 hereby incorporated by reference in their entirety.

[0525] For example, after tumor fragments and / or dissection or digestion of tumor fragments such as those described in step A of FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) under conditions that favor the growth of TILs over tumors and other cells. In some embodiments, IL-2, OKT-3, and feeder cells are added at the start of culture (e.g., on day 0) together with the tumor digest and / or tumor fragments. In some embodiments, the tumor digest and / or tumor fragments are incubated in a container containing up to 60 fragments per container and 6000 IU / mL of IL-2. In some embodiments, this primary cell population is cultured for several days, generally 1 - 8 days, resulting in a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, this primary cell population is cultured for several days, generally 1 - 7 days, resulting in a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, the first priming expansion occurs for 1 - 8 days, resulting in a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, the first priming expansion occurs for 1 - 7 days, resulting in a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, this first priming expansion occurs for 5 - 8 days, resulting in a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, this first priming expansion occurs for 5 - 7 days, resulting in a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, this first priming expansion occurs for 6 - 8 days, resulting in a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, this first priming expansion occurs for 6 - 7 days, resulting in a bulk TIL population, generally about 1×10 8resulting in a bulk of TIL cells. In some embodiments, this first priming expansion occurs over 7 - 8 days, resulting in a bulk TIL population, generally about 1×10 8 resulting in a bulk of TIL cells. In some embodiments, this first priming expansion occurs over about 7 days, resulting in a bulk TIL population, generally about 1×10 8 resulting in a bulk of TIL cells. In some embodiments, this first priming expansion occurs over about 8 days, resulting in a bulk TIL population, generally about 1×10 8 resulting in a bulk of TIL cells.

[0526] In preferred embodiments, the expansion of TILs can be carried out using the first priming expansion step described below and herein (which may include the process referred to as pre - REP or priming REP, and may include feeder cells from day 0 and / or the start of culture, such as those described in step B of FIG. 1 (in particular, for example, FIG. 1B and / or FIG. 1C)), followed by step D below and the second rapid expansion described herein (including the process referred to as the rapid expansion protocol (REP) step), followed by any cryopreservation, and then the second step D described below and herein (including the process referred to as the restimulation REP step). The TILs obtained from this process can optionally be characterized for the phenotypic characteristics and metabolic parameters described herein. In some embodiments, the tumor fragments are about 1 mm 3 to 10 mm 3 in size.

[0527] In some embodiments, the first expansion culture medium is referred to as "CM", an abbreviation for the culture medium. In some embodiments, the CM in step B consists of RPMI 1640 supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin, and contains GlutaMAX .

[0528] In some embodiments, there are 240 or fewer tumor fragments. In some embodiments, there are 240 or fewer tumor fragments placed in 4 or fewer containers. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, 60 or fewer tumor fragments are placed in one container. In some embodiments, each container contains 500 mL or less of medium per container. In some embodiments, the medium contains IL-2. In some embodiments, the medium contains 6000 IU / mL of IL-2. In some embodiments, the medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the medium contains 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains OKT-3. In some embodiments, the medium contains 30 ng / mL of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng of OKT-3, and 2.5×10 8 antigen-presenting feeder cells. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen-presenting feeder cells per container.

[0529] After preparation of the tumor fragments, the resulting cells (i.e., the fragments which are a primary cell population) are cultured in a medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions that favor the growth of TIL over tumors and other cells and that allow for TIL priming and accelerated growth starting from day 0 of culture. In some embodiments, the tumor digest and / or tumor fragments are incubated with 6000 IU / mL of IL-2, as well as antigen-presenting feeder cells and OKT-3. This primary cell population is cultured for several days, generally 1 to 8 days, resulting in a bulk TIL population, generally about 1×10 8resulting in individual bulk TIL cells. In some embodiments, the growth medium during the first priming expansion includes IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for several days, generally 1 to 7 days, resulting in a bulk TIL population, generally about 1×10 8 resulting in individual bulk TIL cells. In some embodiments, the growth medium during the first priming expansion includes IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution has a specific activity of 20 - 30×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a specific activity of 20×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a specific activity of 25×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a specific activity of 30×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 4 - 8×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 5 - 7×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 6×10 6It has a final concentration of IL-2 of IU / mg. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the first priming expansion culture medium contains about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6,000 IU / mL of IL-2, or about 5,000 IU / mL of IL-2. In some embodiments, the first priming expansion culture medium contains from about 9,000 IU / mL to about 5,000 IU / mL of IL-2. In some embodiments, the first priming expansion culture medium contains from about 8,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first priming expansion culture medium contains from about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first priming expansion culture medium contains about 6,000 IU / mL of IL-2. In certain embodiments, the cell culture medium further contains IL-2. In some embodiments, the first priming expansion cell culture medium contains about 3,000 IU / mL of IL-2. In certain embodiments, the first priming expansion cell culture medium further contains IL-2. In preferred embodiments, the first priming expansion cell culture medium contains about 3,000 IU / mL of IL-2. In certain embodiments, the first priming expansion cell culture medium contains about 1,000 IU / mL, about 1,500 IU / mL, about 2,000 IU / mL, about 2,500 IU / mL, about 3,000 IU / mL, about 3,500 IU / mL, about 4,000 IU / mL, about 4,500 IU / mL, about 5,000 IU / mL, about 5,500 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, or about 8,000 IU / mL of IL-2. In certain embodiments, the first priming expansion cell culture medium contains from 1,000 to 2,000 IU / mL, from 2,000 to 3,000 IU / mL, from 3,000 to 4,000 IU / mL, from 4,000 to 5,000 IU / mL, from 5,000 to 6,000 IU / mL, from 6,000 to 7,000 IU / mL, from 7,000 to 8,000 IU / mL, or about 8,000 IU / mL of IL-2.

[0530] In some embodiments, the first priming expansion culture medium comprises IL-15 at about 500 IU / mL, about 400 IU / mL, about 300 IU / mL, about 200 IU / mL, about 180 IU / mL, about 160 IU / mL, about 140 IU / mL, about 120 IU / mL, or about 100 IU / mL. In some embodiments, the first priming expansion culture medium comprises IL-15 from about 500 IU / mL to about 100 IU / mL. In some embodiments, the first priming expansion culture medium comprises IL-15 from about 400 IU / mL to about 100 IU / mL. In some embodiments, the first priming expansion culture medium comprises IL-15 from about 300 IU / mL to about 100 IU / mL. In some embodiments, the first priming expansion culture medium comprises IL-15 at about 200 IU / mL. In some embodiments, the first priming expansion cell culture medium comprises IL-15 at about 180 IU / mL. In certain embodiments, the first priming expansion cell culture medium further comprises IL-15. In preferred embodiments, the first priming expansion cell culture medium comprises IL-15 at about 180 IU / mL.

[0531] In some embodiments, the first priming 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 priming expansion culture medium comprises from about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first priming expansion culture medium comprises from about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first priming expansion culture medium comprises from about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first priming expansion culture medium comprises from about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the first priming expansion culture medium comprises from about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the first priming expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the first priming expansion cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the first priming expansion cell culture medium comprises about 0.5 IU / mL of IL-21. In certain embodiments, the cell culture medium further comprises IL-21. In a preferred embodiment, the first priming expansion cell culture medium comprises about 1 IU / mL of IL-21.

[0532] In some embodiments, the first priming expansion cell culture medium contains OKT-3 antibody. In some embodiments, the first priming expansion cell culture medium contains about 30 ng / mL of OKT-3 antibody. In some embodiments, the first priming expansion cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT-3 antibody. In some embodiments, the cell culture medium contains 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, and 50 ng / mL to 100 ng / mL. In some embodiments, the cell culture medium contains OKT-3 antibody at a concentration of 15 ng / mL to 30 ng / mL. In some embodiments, the cell culture medium contains 30 ng / mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

Table 3

[0533] In some embodiments, the first priming expansion 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 in the cell culture medium of from 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of from 20 μg / mL to 40 μg / mL.

[0534] In some embodiments, in addition to one or more TNFRSF agonists, the first priming expansion cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the first priming expansion cell culture medium further comprises IL-2 at an initial concentration of about 6000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and one or more TNFRSF agonists comprise a 4-1BB agonist.

[0535] In some embodiments, the first priming 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, CM consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In some embodiments, CM is CM1 as described in the examples; see Example A. In some embodiments, the first priming expansion occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the first priming expansion culture medium or the initial cell culture medium or the first cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (also referred to herein as feeder cells).

[0536] In some embodiments, the medium used in the expansion process disclosed herein is a serum-free medium or a synthetic medium. In some embodiments, the serum-free or synthetic medium comprises a basal cell medium as well as a serum supplement and / or a serum replacement. In some embodiments, the serum-free or synthetic medium is used to prevent and / or reduce experimental variations that are due in part to variations between lots of serum-containing media.

[0537] In some embodiments, the serum-free or synthetic medium comprises a basal cell medium and a serum supplement and / or a serum replacement. In some embodiments, the basal cell medium includes, but is not limited to, CTS(™) OpTmizer(™) T-cell Expansion Basal Medium, CTS(™) OpTmizer(™) T-Cell Expansion SFM, CTS(™) AIM-V Medium, CTS(™) AIM-V SFM, LymphoONE(™) T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0538] In some embodiments, the serum supplement or serum replacement includes, but is not limited to, CTS(™) OpTmizer T-Cell Expansion Serum Supplement, CTS(™) Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the synthetic medium comprises albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element portion Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3” , Ge4+ 、Se 4+ 、Br, T, Mn 2+ 、P, Si 4+ 、V 5+ 、Mo 6+ 、Ni 2+ 、Rb + 、Sn 2+ 、and Zr 4+ It includes one or more components selected from the group consisting of compounds containing

[0539] In some embodiments, CTS(™) OpTmizer(™) T-cell Immune Cell Serum Replacement is used together with conventional growth media including, but not limited to, CTS(™) OpTmizer(™) T-cell Expansion Basal Medium, CTS(™) OpTmizer(™) T-cell Expansion SFM, C TS(™) AIM-V Medium, CST(™) AIM-V SFM, LymphoONE(™) T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0540] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or synthetic medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or synthetic medium.

[0541] In some embodiments, the serum-free or synthetic medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion The SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0542] In some embodiments, the synthetic medium is CTS™ OpTmizer™ T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T-cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T-cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, the CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (Thermo supplemented with Fisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains from about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 3000 IU / mL of IL-2. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 6000 IU / mL of IL-2. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains about 3000 IU / mL of IL-2.In some embodiments, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS(Trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from approximately 1000 IU / mL to approximately 6000 IU / mL of IL-2. In some embodiments, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS(Trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and further contains from approximately 1000 IU / mL to approximately 8000 IU / mL of IL-2. In some embodiments, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS(Trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and further contains approximately 3000 IU / mL of IL-2. In some embodiments, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS(Trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and further contains approximately 6000 IU / mL of IL-2. In some embodiments, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS(Trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0543] In some embodiments, the serum-free medium or synthetic medium contains from approximately 0.1 mM to approximately 10 mM, 0.5 mM to approximately 9 mM, 1 mM to approximately 8 mM, 2 mM to approximately 7 mM, 3 mM to approximately 6 mM, or or glutamine (i.e., GlutaMAX™) is supplemented at a concentration of 4 mM to about 5 mM. In some embodiments, the serum-free medium or synthetic medium is supplemented with glutamine (i.e., GlutaMAX™) at a concentration of about 2 mM.

[0544] In some embodiments, the serum-free medium or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0545] In some embodiments, the synthetic medium described in International PCT Publication No. WO / 1998 / 030679, which is incorporated herein by reference, is useful in the present invention. That publication describes a serum-free eukaryotic cell culture medium. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth under serum-free culture. The serum-free eukaryotic cell culture medium supplement includes one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics, or is obtained by combining them. In some embodiments, the synthetic medium further includes L-glutamine, sodium bicarbonate, and / or β-mercaptoethanol. In some embodiments, the synthetic medium includes an albumin or albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the synthetic medium includes albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag + 、Al 3+ 、Ba 2+ 、Cd 2+ 、Co 2+ 、Cr 3” 、Ge 4+ 、Se 4+ 、Br、T、Mn 2+ 、P、Si 4+ 、V 5+ 、Mo 6+ 、Ni 2+, Rb + , Sn 2+ , and Zr 4+ It includes one or more components selected from the group consisting of compounds containing. In some embodiments, the basal cell medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco Medium.

[0546] In some embodiments, the concentration of glycine in the synthetic medium ranges from about 5 to 200 mg / L, the concentration of L-histidine is about 5 to 250 mg / L, the concentration of L-isoleucine is about 5 to 300 mg / L, the concentration of L-methionine is about 5 to 200 mg / L, the concentration of L-phenylalanine is about 5 to 400 mg / L, the concentration of L-proline is about 1 to 1000 mg / L, the concentration of L-hydroxyproline is about 1 to 45 mg / L, the concentration of L-serine is about 1 to 250 mg / L, the concentration of L-threonine is about 10 to 500 mg / L, the concentration of L-tryptophan is about 2 to 110 mg / L, the concentration of L-tyrosine is about 3 to 175 mg / L, the concentration of L-valine is about 5 to 500 mg / L, the concentration of thiamine is about 1 to 20 mg / L, reduced the concentration of glutathione is about 1 to 20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg / L, the concentration of iron-saturated transferrin is about 1 to 50 mg / L, the concentration of insulin is about 1 to 100 mg / L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5000 to 50,000 mg / L.

[0547] In some embodiments, the non-trace element sub-components in the synthetic medium are present in the concentration ranges listed in the column under the heading "Concentration Range in 1× Medium" in Table A below. In other embodiments, the non-trace element sub-components in the synthetic medium are present at the final concentrations listed in the column under the heading "Preferred Embodiments of 1× Medium" in Table A below. In other embodiments, the synthetic medium is a basal cell medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-trace element sub-components of the types and concentrations listed in the column under the heading "Preferred Embodiments in Supplement" in Table A below.

Table 4

[0548] In some embodiments, the osmotic pressure of the synthetic medium is about 260 - 350 mOsmol. In some embodiments, the osmotic pressure is about 280 - 310 mOsmol. In some embodiments, the synthetic medium is supplemented with up to about 3.7 g / L, or about 2.2 g / L of sodium bicarbonate. The synthetic medium may further be supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0549] In some embodiments, the synthetic medium described in Smith, et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement," Clin Transl Immunology, 4(1) 2015 (doi:10.1038 / cti.2014.31) is useful in the present invention. Briefly, RPMI or CTS™ OpTmizer was used as the basal cell medium and supplemented with 0, 2%, 5%, or 10% of CTS™ Immune Cell Serum Replacement.

[0550] In certain embodiments, the cell culture medium within the first and / or second gas permeable containers is not filtered. The use of unfiltered cell culture medium can simplify the procedures necessary to expand the number of cells. In certain embodiments, the cell culture medium within the first and / or second gas permeable containers lacks β-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

[0551] In some embodiments, the first priming expansion (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is, as considered in the examples and figures, from 1 to 8 days. In some embodiments, the first priming expansion (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is from 2 to 8 days. In some embodiments, the first priming expansion (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is from 3 to 8 days. In some embodiments, the first priming expansion (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is, as considered in the examples and figures, from 4 to 8 days. In some embodiments, the first priming expansion (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is, as considered in the examples and figures, from 1 to 7 days. In some embodiments, the first priming expansion (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is from 2 to 8 days. In some embodiments, the first priming expansion (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is from 2 to 7 days.In some embodiments, the first priming expansion (including, for example, processes such as those described in step B of FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), which may include those that may also be referred to as pre-REP or priming REP) process is 3 to 8 days. In some embodiments, the first priming expansion (including, for example, processes such as those described in step B of FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), which is referred to as pre-REP or priming REP. The (process that may also include things that may be) process is 3 to 7 days. In some embodiments, the first priming extension (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may also include things that may be referred to as pre-REP or priming REP) process is 4 to 8 days. In some embodiments, the first priming extension (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may also include things that may be referred to as pre-REP or priming REP) process is 4 to 7 days. In some embodiments, the first priming extension (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may also include things that may be referred to as pre-REP or priming REP) process is 5 to 8 days. In some embodiments, the first priming extension (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may also include things that may be referred to as pre-REP or priming REP) process is 5 to 7 days. In some embodiments, the first priming extension (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may also include things that may be referred to as pre-REP or priming REP) process is 6 to 8 days. In some embodiments, the first priming extension (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may also include things that may be referred to as pre-REP or priming REP) process is 6 to 7 days. In some embodiments, the first priming extension (including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), which may also include things that may be referred to as pre-REP or priming REP) process is 7 to 8 days.In some embodiments, the first priming extension (e.g., including the process such as that provided in step B of FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), which may include what may also be referred to as pre-REP or priming REP) process is 8 days. In some embodiments, the first priming extension (e.g., including the process such as that described in step B of FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), which may include what may also be referred to as pre-REP or priming REP) process is 7 days.

[0552] In some embodiments, the first priming TIL expansion can continue for 1 to 8 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 1 to 7 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 2 to 8 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 2 to 7 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 3 to 8 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 3 to 7 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 4 to 8 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 4 to 7 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion starts when fragmentation occurs and / or when the first priming expansion step is started It can continue for 5 to 8 days from when it is carried out. In some embodiments, the first priming TIL expansion can continue for 5 to 7 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 6 to 8 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 6 to 7 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 7 to 8 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 8 days from when fragmentation occurred and / or when the first priming expansion step was initiated. In some embodiments, the first priming TIL expansion can continue for 7 days from when fragmentation occurred and / or when the first priming expansion step was initiated.

[0553] In some embodiments, the first priming expansion of TILs can last for 1, 2, 3, 4, 5, 6, 7, or 8 days. In some embodiments, the first TIL expansion can last from 1 to 8 days. In some embodiments, the first TIL expansion can last from 1 to 7 days. In some embodiments, the first TIL expansion can last from 2 to 8 days. In some embodiments, the first TIL expansion can last from 2 to 7 days. In some embodiments, the first TIL expansion can last from 3 to 8 days. In some embodiments, the first TIL expansion can last from 3 to 7 days. In some embodiments, the first TIL expansion can last from 4 to 8 days. In some embodiments, the first TIL expansion can last from 4 to 7 days. In some embodiments, the first TIL expansion can last from 5 to 8 days. In some embodiments, the first TIL expansion can last from 5 to 7 days. In some embodiments, the first TIL expansion can last from 6 to 8 days. In some embodiments, the first TIL expansion can last from 6 to 7 days. In some embodiments, the first TIL expansion can last from 7 to 8 days. In some embodiments, the first TIL expansion can last for 8 days. In some embodiments, the first TIL expansion can last for 7 days.

[0554] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the first priming expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, can be included during the first expansion, for example, according to FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), and including during the step B process described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first priming expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, can be included according to FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), and during the step B process described herein.

[0555] In some embodiments, the first priming expansion, for example, step B according to FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), is performed in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL expansion. In some embodiments, a bioreactor is used. In some embodiments, the bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

[0556] 1. Feeder cells and antigen-presenting cells In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL expansion, rather, they are added during the first priming expansion at any point between days 4 - 8. In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP)) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL expansion, rather, they are added during the first priming expansion at any point between days 4 - 7. In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP)) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL expansion, rather, they are added during the first priming expansion at any point between days 5 - 8. In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP)) does not require feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL expansion, rather, they are added during the first priming expansion at any point between days 5 - 7.In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL expansion. Rather, they are added during the first priming expansion at any point on days 6 - 8. In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL expansion. Rather, they are added during the first priming expansion at any point on days 6 - 7. In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL expansion. Rather, they are added during the first priming expansion at any point on day 7 or 8. In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL expansion. Rather, they are added during the first priming expansion at any point on day 7. In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL expansion. Rather, they are added during the first priming expansion at any point on day 8. and including expansions such as those referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of TIL expansion. Rather, they are added during the first priming expansion at any point on day 8.

[0557] In certain embodiments, the first priming expansion procedure described herein (e.g., as described in step B from FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), and including expansions such as those referred to as pre-REP or priming REP) requires feeder cells (also referred to herein as “antigen-presenting cells”) at the start of TIL expansion and during the first priming expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard unit of whole blood from a healthy allogeneic donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In some embodiments, 2.5×10 8 feeder cells are used during the first priming expansion. In some embodiments, 2.5×10 8 feeder cells per vessel are used during the first priming expansion. In some embodiments, 2.5×10 8 feeder cells per GREX-10 are used during the first priming expansion. In some embodiments, 2.5×10 8 feeder cells per GREX-100 are used during the first priming expansion.

[0558] Generally, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the REP procedure as described in the examples, which provides an exemplary protocol for assessing the non-replicative ability of irradiated allogeneic PBMCs.

[0559] In some embodiments, if the total viable cell count on day 14 is less than the initial viable cell count cultured on day 0 of the first priming expansion, the PBMCs are considered to be non-replicative and are approved for use in the TIL expansion procedure described herein.

[0560] In some embodiments, if the total viable cell count cultured in the presence of OKT3 and IL-2 on day 7 did not increase from the initial viable cell count cultured on day 0 of the first priming expansion, the PBMCs are considered to have no replication ability and are approved for use in the TIL expansion procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 3000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0561] In some embodiments, if the total viable cell count cultured in the presence of OKT3 and IL-2 on day 7 did not increase from the initial viable cell count cultured on day 0 of the first priming expansion, the PBMCs are considered to have no replication ability and are approved for use in the TIL expansion procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 5 - 60 ng / mL of OKT3 antibody and 1000 - 6000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 10 - 50 ng / mL of OKT3 antibody and 2000 - 5000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 20 - 40 ng / mL of OKT3 antibody and 2000 - 4000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 25 - 35 ng / mL of OKT3 antibody and 2500 - 3500 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 15 ng / mL of OKT3 antibody and 3000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 15 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0562] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In certain embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion 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 certain embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is from 1:50 to 1:300. In certain embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is from 1:100 to 1:200.

[0563] In certain embodiments, the first priming expansion procedure described herein requires about 2.5×10 8 feeder cells: about 100×10 6 TILs ratio. In another embodiment, the first priming expansion procedure described herein requires about 2.5×10 8 feeder cells: about 50×10 6 TILs ratio. In yet another embodiment, the first priming expansion procedure described herein requires about 2.5×10 8 feeder cells: about 25×10 6 TILs. In yet another embodiment, the first priming expansion described herein requires about 2.5×10 8 feeder cells. In yet another embodiment, the first priming expansion requires one-fourth, one-third, five-twelfths, or one-half of the number of feeder cells used in the second rapid expansion.

[0564] In some embodiments, the medium in the first priming expansion contains IL-2. In some embodiments, the medium in the first priming expansion contains 6000 IU / mL of IL-2. In some embodiments, the medium in the first priming expansion contains antigen-presenting feeder cells. In some embodiments, the medium in the first priming expansion contains 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium in the first priming expansion contains OKT-3. In some embodiments, the medium contains 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen-presenting feeder cells. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains 500 mL of culture medium per container and 15 μg of OKT-3 per 2.5×10 8 antigen-presenting feeder cells. In some embodiments, the medium contains 500 mL of culture medium per container and 15 μg of OKT-3. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 500 mL of culture medium, 6000 IU / mL of IL-2, 30 ng / mL ng of OKT-3, and 2.5×10 8 antigen-presenting feeder cells. In some embodiments, the medium contains 500 mL of culture medium, 6000 IU / mL of IL-2, 15 μg of OKT-3, and 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains 500 mL of culture medium per container and 15 μg of OKT-3 per 2.5×10 8 antigen-presenting feeder cells.

[0565] In certain embodiments, the first priming expansion procedure described herein requires an excess of feeder cells beyond the TIL during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard unit of whole blood from a healthy allogeneic donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In certain embodiments, artificial antigen presenting (aAPC) cells are used in place of PBMCs.

[0566] Generally, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.

[0567] In certain embodiments, artificial antigen presenting cells are used in the first priming expansion as an alternative to PBMCs or in combination with PBMCs.

[0568] 2. Cytokines The expansion methods described herein generally use a culture medium containing high doses of cytokines, particularly IL-2, as is known in the art.

[0569] Alternatively, as generally outlined in International Publication Nos. WO2015 / 189356 and WO2015 / 189357, which are hereby expressly incorporated by reference in their entirety, it is further possible to use a combination of cytokines for the first priming expansion of TILs in a combination of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter being particularly useful in many embodiments. The use of cytokine combinations works particularly advantageously for the generation of lymphocytes, particularly T cells as described therein.

Table 5

[0570] C. Step C: Transition from the First Priming Expansion to the Second Rapid Expansion In some cases, for example, the bulk TIL population obtained from the first priming expansion (which may include an expansion sometimes referred to as pre-REP) containing the TIL population obtained from step B as shown in FIG. 1 (in particular, for example, FIGS. 1B and / or 1C) can be subjected to a second rapid expansion (which may include an expansion sometimes referred to as the rapid expansion protocol (REP)) and then cryopreserved as discussed below. Similarly, when genetically modified TILs are used in therapy, the expanded TIL population from the first priming expansion or the expanded TIL population from the second rapid expansion can be subjected to genetic modification for suitable therapy before the expansion step or after the first priming expansion and before the second rapid expansion.

[0571] In some embodiments, the TILs obtained from the first priming expansion (e.g., from step B as shown in FIG. 1 (in particular, for example, FIGS. 1B and / or 1C)) are stored until they are phenotyped for selection. In some embodiments, the TILs obtained from the first priming expansion (e.g., from step B as shown in FIG. 1 (in particular, for example, FIGS. 1B and / or 1C)) are not stored and proceed directly to the second rapid expansion. In some embodiments, the TILs obtained from the first priming expansion are not cryopreserved after the first priming expansion and before the second rapid expansion. In some embodiments, the transition from the first priming expansion to the second expansion occurs when tumor fragmentation occurs and / or the first p It occurs approximately 2, 3, 4, 5, 6, 7, or 8 days after the priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs when fragmentation occurs and / or approximately 3 to 7 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs when fragmentation occurs and / or approximately 3 to 8 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 4 to 7 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 4 to 8 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 5 to 7 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 5 to 8 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 6 to 7 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 6 to 8 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 7 to 8 days after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs when fragmentation occurs and / or approximately 7 days after the first priming expansion step is initiated.In some embodiments, the transition from the first priming expansion to the second expansion occurs at the time fragmentation occurred and / or about 8 days from when the first priming expansion step was initiated.

[0572] In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 1, 2, 3, 4, 5, 6, 7, or 8 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 1 to 7 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 1 to 8 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs 2 to 7 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs 2 to 8 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs 3 to 7 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second expansion occurs 3 to 8 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 4 to 7 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 4 to 8 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 5 to 7 days after fragmentation occurs and / or after the first priming expansion step is initiated. In some embodiments In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 5 to 8 days after fragmentation occurred and / or after the first priming expansion step was initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 6 to 7 days after fragmentation occurred and / or after the first priming expansion step was initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 6 to 8 days after fragmentation occurred and / or after the first priming expansion step was initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 7 to 8 days after fragmentation occurred and / or after the first priming expansion step was initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 7 days after fragmentation occurred and / or after the first priming expansion step was initiated. In some embodiments, the transition from the first priming expansion to the second rapid expansion occurs 8 days after fragmentation occurred and / or after the first priming expansion step was initiated.

[0573] In some embodiments, the TILs are not stored after the first initial expansion and before the second rapid expansion, and the TILs proceed directly to the second rapid expansion (e.g., in some embodiments, not stored during the transition from step B to step D as shown in FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C)). In some embodiments, the transition occurs within a closed system as described herein. In some embodiments, the second TIL population, which is TILs from the first priming expansion, proceeds directly to the second rapid expansion without a transition period.

[0574] In some embodiments, the transition from the first priming expansion to the second rapid expansion, e.g., step C according to FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C), is performed within a closed - system bioreactor. In some embodiments, as described herein, a closed - system is used for TIL expansion. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, GREX - 10 or GREX - 100. In some embodiments, the closed - system bioreactor is a single bioreactor. In some embodiments, the transition from the first priming expansion to the second rapid expansion involves a scale - up of the vessel size. In some embodiments, the first priming expansion is performed in a vessel smaller than the second rapid expansion. In some embodiments, the first priming expansion is performed within GREX - 100 and the second rapid expansion is performed within GREX - 500.

[0575] D. Step D: Second rapid expansion In some embodiments, the TIL cell population is further expanded after collection and the first priming expansion, after steps A and B and the transition referred to as step C as shown in FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C). This further expansion is referred to herein as the second rapid expansion, which may include an expansion process generally referred to in the art as a rapid expansion process (rapid expansion protocol or REP, and the process shown in step D of FIG. 1 (in particular, e.g., FIGS. 1B and / or 1C)). The second rapid expansion is generally achieved within a gas - permeable container using a culture medium containing several components, including feeder cells, cytokine source, and anti - CD3 antibody. In some embodiments, 1, 2, 3, or 4 days after the start of the second rapid expansion (i.e., on the 8th, 9th, 10th, or 11th day of the overall Gen3 process), the TILs are transferred to a larger - volume container.

[0576] In some embodiments, the second rapid expansion of TIL (also referred to as REP and may include the process shown in step D of FIG. 1, particularly, for example, FIGS. 1B and / or 1C) may use any TIL flask or container known to those skilled in the art. It can be carried out. In some embodiments, the second TIL expansion can continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 1 day to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 1 day to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 2 days to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 2 days to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 3 days to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 3 days to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 4 days to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 4 days to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 5 days to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 5 days to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 6 days to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 6 days to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 7 days to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 7 days to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 8 days to about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 8 days to about 10 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can continue for about 9 days to about 10 days after the start of the second rapid expansion.In some embodiments, the second TIL expansion can last for about 1 day after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 2 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 3 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 4 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 5 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 6 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 7 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 8 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 9 days after the start of the second rapid expansion. In some embodiments, the second TIL expansion can last for about 10 days after the start of the second rapid expansion.

[0577] In one embodiment, the second rapid expansion can be performed in a gas-permeable container using the methods of the present disclosure (including, for example, an expansion referred to as REP and the process shown in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)). In some embodiments, the TILs are expanded in the second rapid expansion in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the TILs are expanded in the second rapid expansion in the presence of IL-2, OKT-3, and feeder cells, and the feeder cells are added to a final concentration that is 2-fold, 2.4-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold the concentration of the feeder cells present in the first priming expansion. For example, the TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulants include, for example, anti-CD3 antibodies, such as Alternatively, it may contain about 30 ng / mL of OKT3, a murine monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA), or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further stimulation of TILs in vitro by including one or more antigens during a second expansion, including antigenic portions such as cancer epitope(s), which can be optionally expressed in the presence of a T cell growth factor such as 300 IU / mL of IL-2 or IL-15 from a vector, such as a human leukocyte antigen A2 (HLA-A2)-binding peptide, such as 0.3 μM MART-1:26-35 (27L) or gpl00:209-217 (210M). Other suitable antigens can 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 cancer antigen(s) pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, TILs can be further restimulated, for example, with irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2 as an example. In some embodiments, the restimulation occurs as part of the second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or using irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0578] In certain embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In certain embodiments, 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 certain embodiments, 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 8000 IU / mL of IL-2.

[0579] In certain embodiments, the cell culture medium contains OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng / mL of OKT-3 antibody. In certain embodiments, the cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of OKT-3 antibody. In certain embodiments, the cell culture medium contains OKT-3 antibody at 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, and 50 ng / mL to 100 ng / mL. In certain embodiments, the cell culture medium contains about 30 ng / mL to 60 ng / mL of OKT-3 antibody. In certain embodiments, the cell culture medium contains about 60 ng / mL of OKT-3. In some embodiments, the OKT-3 antibody is muromonab.

[0580] In some embodiments, the medium in the second rapid expansion contains IL-2. In some embodiments, the medium contains 6000 IU / mL of IL-2. In some embodiments, the medium in the second rapid expansion contains antigen-presenting feeder cells. In some embodiments the medium in the second rapid expansion contains 7.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium in the second rapid expansion contains OKT-3. In some embodiments, the second rapid expansion medium contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the second rapid expansion medium contains 6000 IU / mL of IL-2, 60 ng / mL of OKT-3, and 7.5×10 8contains individual antigen-presenting feeder cells. In some embodiments, the medium is 500 mL of culture medium per container, 6000 IU / mL of IL-2, 30 μg of OKT-3, and 7.5×10 8 contains individual antigen-presenting feeder cells.

[0581] In some embodiments, the medium in the second rapid expansion contains IL-2. In some embodiments, the medium contains 6000 IU / mL of IL-2. In some embodiments, the medium in the second rapid expansion contains antigen-presenting feeder cells. In some embodiments, the medium is 5×10 8 ~7.5×10 8 individual antigen-presenting feeder cells per container. In some embodiments, the medium in the second rapid expansion contains OKT-3. In some embodiments, the medium in the second rapid expansion contains 500 mL of culture medium per container and 30 μg of OKT-3. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium in the second rapid expansion contains 6000 IU / mL of IL-2, 60 ng / mL of OKT-3, and 5×10 8 ~7.5×10 8 individual antigen-presenting feeder cells. In some embodiments, the medium in the second rapid expansion is 500 mL of culture medium per container, as well as 6000 IU / mL of IL-2, 30 μg of OKT-3, and 5×10 8 ~7.5×10 8 individual antigen-presenting feeder cells.

[0582] 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 in the cell culture medium of from 0.1 μg / mL to 100 μg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of from 20 μg / mL to 40 μg / mL.

[0583] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0584] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and any combination thereof, may be included during the second expansion, for example, according to FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), and including during the step D process described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included according to FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), and during the step D process described herein.

[0585] In some embodiments, the second expansion can 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 occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs, also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0586] In some embodiments, the second expansion culture medium comprises about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises from about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises from about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises from 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 certain embodiments, the cell culture medium further comprises IL-15. In preferred embodiments, the cell culture medium comprises about 180 IU / mL of IL-15.

[0587] 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 from 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 from 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 from about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises from about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises from about 5 IU / mL of IL-21 to 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 certain embodiments, the cell culture medium further comprises IL-21. In preferred embodiments, the cell culture medium comprises about 1 IU / mL of IL-21.

[0588] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In certain embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells in rapid expansion and / or second expansion is about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:75, 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 certain embodiments, the ratio of TILs to PBMCs in rapid expansion and / or second expansion is from 1:50 to 1:300. In certain embodiments, the ratio of TILs to PBMCs in rapid expansion and / or second expansion is from 1:100 to 1:200.

[0589] In certain embodiments, REP and / or second rapid expansion is performed in a flask in which bulk TILs are mixed with 100-fold or 200-fold excess of inactivated feeder cells, 30 ng / mL of OKT3, an anti-CD3 antibody, and 6000 IU / mL of IL-2 in 150 mL of medium, and the feeder cell concentration is at least 1.1 times (1.1X), 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.8 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, or 4.0 times the feeder cell concentration in the first priming expansion. Medium exchanges are performed until the cells are transferred to an alternative growth chamber (generally, a two-thirds medium exchange by aspiration of two-thirds of the used medium and replacement with an equal volume of fresh medium). Alternative growth chambers include G-REX flasks and gas permeable containers, as more fully contemplated below.

[0590] In some embodiments, the second rapid expansion (which may include the process referred to as the REP process) is 7 to 9 days, as discussed in the examples and figures. In some embodiments, the second expansion is 7 days. In some embodiments, the second expansion is 8 days. In some embodiments, the second expansion is 9 days.

[0591] In one embodiment, the second expansion (which may include the expansion referred to as REP, as well as the expansion referenced in step D of FIG. 1 (in particular, for example, FIGS. 1B and / or 1C)) can be performed in a 500 mL capacity gas permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) having a 100 cm gas permeable silicon bottom. 5×10 6 or 10×10 6 individual TILs can be cultured with PBMCs in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL of IL-2, and 30 ng / mL of anti-CD3 (OKT3). The G-Rex 100 flask can be incubated at 37° C. in 5% CO 2 2. On day 5, 250 mL of the supernatant is removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum, 6000 IU / mL of IL-2, and returned to and added to the original GREX-100 flask. If the TILs are continuously expanded in the GREX-100 flask, on day 10 or 11, the TILs can be transferred to a larger flask such as a GREX-500. On day 14 of culture, the cells can be harvested. On day 15 of culture, the cells can be harvested. On day 16 of culture, the cells can be harvested. In some embodiments, the media exchange is performed until the cells are transferred to an alternative growth chamber. In some embodiments, two-thirds of the media is exchanged by aspiration of the used media and replacement with an equal volume of fresh media. In some embodiments, alternative growth chambers include GREX flasks and gas permeable containers, as more fully discussed below.

[0592] In some embodiments, the medium used in the expansion processes disclosed herein is a serum-free medium or a synthetic medium. In some embodiments, the serum-free or synthetic medium comprises a basal cell medium as well as a serum supplement and / or a serum replacement. In some embodiments, the serum-free or synthetic medium is used to prevent and / or reduce experimental variability that is due in part to variability between lots of serum-containing media.

[0593] In some embodiments, the serum-free or synthetic medium comprises a basal cell medium as well as a serum supplement and / or a serum replacement. In some embodiments, the basal cell medium includes, but is not limited to, CTS™ OpTmizer™ T-cell Expansion Basal Medium, CTS™ OpTmizer™ T-Cell Expan sion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0594] In some embodiments, the serum supplement or serum replacement includes, but is not limited to, one or more of CTS(™) OpTmizer T-Cell Expansion Serum Supplement, CTS(™) Immune Cell Serum Replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the synthetic medium includes albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3” , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ and includes one or more components selected from the group consisting of compounds containing the same. In some embodiments, the synthetic medium further includes L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0595] In some embodiments, CTS(Trademark) OpTmizer(Trademark) T-cell Immune Cell Serum Replacement is used with conventional growth media including, but not limited to, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion Basal Medium, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM, CTS(Trademark) AIM-V Medium, CST(Trademark) AIM-V SFM, LymphoONE(Trademark) T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0596] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or synthetic medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or synthetic medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or synthetic medium.

[0597] In some embodiments, the serum-free or synthetic medium is CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS(Trademark) OpTmizer(Trademark) is useful in the present invention. CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM is 1L of CTS(Trademark) OpTmizer(Trademark) T-c ell Expansion Basal Medium, in combination with 26 mL of CTS™ OpTmizer™ T-Cell Expansion Supplement, which are mixed together prior to use. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0598] In some embodiments, the synthetic medium is CTS(™) OpTmizer(™) T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS(™) OpTmizer(™) is useful in the present invention. CTS(™) OpTmizer(™) T-cell Expansion SFM is a combination of 1 L of CTS(™) OpTmizer(™) T-cell Expansion Basal Medium and 26 mL of CTS(™) OpTmizer(™) T-Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with about 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with about 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS(™) OpTmizer(™) T-cell Expansion SFM is supplemented with about 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 1000 IU / mL to about 8000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains approximately 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains approximately 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T-cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from approximately 1000 IU / mL to approximately 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTm. The izer (trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS (trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains approximately 3000 IU / mL of IL-2. In some embodiments, the CTS (trademark) OpTmizer (trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS (trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from approximately 1000 IU / mL to approximately 6000 IU / mL of IL-2. In some embodiments, the CTS (trademark) OpTmizer (trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS (trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and further contains from approximately 1000 IU / mL to approximately 8000 IU / mL of IL-2. In some embodiments, the CTS (trademark) OpTmizer (trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS (trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and further contains approximately 3000 IU / mL of IL-2. In some embodiments, the CTS (trademark) OpTmizer (trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS (trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and approximately 2 mM glutamine, and further contains approximately 6000 IU / mL of IL-2.In some embodiments, CTS(Trademark) OpTmizer(Trademark) T-cell Expansion SFM is supplemented with approximately 3% CTS(Trademark) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0599] In some embodiments, the serum-free medium or synthetic medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or synthetic medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0600] In some embodiments, the serum-free medium or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or synthetic medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

[0601] In some embodiments, the synthetic medium described in International PCT Publication No. WO / 1998 / 030679, which is incorporated herein by reference, is useful in the present invention. That publication describes a serum-free eukaryotic cell culture medium. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth under serum-free culture. The serum-free eukaryotic cell culture medium supplement includes one or more albumin or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics, or is obtained by combining them In some embodiments, the synthetic medium further includes L-glutamine, sodium bicarbonate, and / or β-mercaptoethanol. In some embodiments, the synthetic medium includes albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the synthetic medium includes albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element portion Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3” , Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni2+ , Rb + , Sn 2+ , and Zr 4+ It includes one or more components selected from the group consisting of compounds containing. In some embodiments, the basal cell medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco Medium.

[0602] In some embodiments, the concentration of glycine in the synthetic medium ranges from about 5 to 200 mg / L, the concentration of L-histidine is about 5 to 250 mg / L, the concentration of L-isoleucine is about 5 to 300 mg / L, the concentration of L-methionine is about 5 to 200 mg / L, the concentration of L-phenylalanine is about 5 to 400 mg / L, the concentration of L-proline is about 1 to 1000 mg / L, the concentration of L-hydroxyproline is about 1 to 45 mg / L, the concentration of L-serine is about 1 to 250 mg / L, the concentration of L-threonine is about 10 to 500 mg / L, the concentration of L-tryptophan is about 2 to 110 mg / L, the concentration of L-tyrosine is about 3 to 175 mg / L, the concentration of L-valine is about 5 to 500 mg / L, the concentration of thiamine is about 1 to 20 mg / L, the concentration of reduced glutathione is about 1 to 20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg / L, the concentration of iron-saturated transferrin is about 1 to 50 mg / L, the concentration of insulin is about 1 to 100 mg / L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5000 to 50,000 mg / L.

[0603] In some embodiments, the non-trace element sub-components in the synthetic medium are present in the concentration ranges listed in the column under the heading "Concentration Range in 1× Medium" of Table A below. In other embodiments, the non-trace element sub-components in the synthetic medium are present at the final concentrations listed in the column under the heading "Preferred Embodiments of 1× Medium" of Table A below. In other embodiments, the synthetic medium is a basal cell medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-trace element sub-components of the types and concentrations listed in the column under the heading "Preferred Embodiments in Supplement" of Table A below. [Table 6]

[0604] In some embodiments, the osmotic pressure of the synthetic medium is about 260 - 350 mOsmol. In some embodiments, the osmotic pressure is about 280 - 310 mOsmol. In some embodiments, the synthetic medium is supplemented with up to about 3.7 g / L, or about 2.2 g / L of sodium bicarbonate. The synthetic medium may further be supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0605] In some embodiments, the synthetic medium described in Smith, et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement," Clin Transl Immunology, 4(1) 2015 (doi:10.1038 / cti.2014.31) is useful in the present invention. Briefly, RPMI or CTS™ OpTmizer was used as the basal cell medium and supplemented with 0, 2%, 5%, or 10% of CTS™ Immune Cell Serum Replacement.

[0606] In certain embodiments, the cell culture medium within the first and / or second gas permeable containers is not filtered. The use of unfiltered cell culture medium can simplify the procedures necessary to expand the number of cells. In certain embodiments, the cell culture medium within the first and / or second gas permeable containers lacks β-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

[0607] In certain embodiments, a second rapid expansion (including the expansion referred to as REP) is performed, further comprising the step of selecting TILs for their superior tumor reactivity. Any selection method known in the art may 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, may be used for the selection of TILs for their superior tumor reactivity.

[0608] Optionally, the cell viability assay can be performed after the second rapid expansion (including the expansion referred to as REP expansion) using standard assays known in the art. For example, a trypan blue exclusion assay that selectively labels dead cells and enables the evaluation of viability can be performed on samples of bulk TILs. In some embodiments, the TIL samples can be counted using a Cellometer K2 Automated Cell Counter (Nexcelom Bioscience, Lawrence, MA), and the viability can be determined. In some embodiments, the viability is determined according to the standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.

[0609] The diverse antigen receptors of T lymphocytes and B lymphocytes are produced by somatic recombination of a limited number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides a method for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, the TILs obtained by this method exhibit an increase in T cell repertoire diversity. In some embodiments, the TILs obtained in the second expansion exhibit an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the diversity is in the immunoglobulin and in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin and in the immunoglobulin light chain. In some embodiments, the diversity is in the T cell receptor. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and / or β is increased. In some embodiments, the expression of T cell receptor (TCR) α is increased. In some embodiments, the expression of T cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα / β) is increased.

[0610] In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as will be discussed in more detail below. In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains, as will be discussed in more detail below, 6000 IU / mL of IL-2, 30 μg of OKT-3 per flask, and 7.5×10 8It contains individual antigen-presenting feeder cells (APCs). In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains IL-2, OKT-3, as well as antigen-presenting feeder cells (APCs), as will be discussed in more detail below. In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains 6000 IU / mL of IL-2, 30 μg of OKT-3 per flask, and 5×10 8 individual antigen-presenting feeder cells (APCs).

[0611] In some embodiments, the second rapid expansion, e.g., step D according to FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C), is performed in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL expansion. In some embodiments, a bioreactor is used. In some embodiments, the bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-100 or G-REX-500. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-500.

[0612] 1. Feeder Cells and Antigen-Presenting Cells In certain embodiments, the second rapid expansion procedure described herein (e.g., the expansion as described in step D of FIG. 1 (particularly, e.g., FIGS. 1B and / or 1C), and including what is referred to as REP) requires an excess of feeder cells during REP TIL expansion and / or during the second rapid expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

[0613] In general, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the REP procedure as described in the examples, which provides an exemplary protocol for assessing the non - replicative ability of irradiated allogeneic PBMCs.

[0614] In some embodiments, if the total viable cell count on day 7 or day 14 is less than the initial viable cell count cultured on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to have no replicative ability and are approved for use in the TIL expansion procedure described herein.

[0615] In some embodiments, if the total viable cell count cultured in the presence of OKT3 and IL - 2 on day 7 and day 14 did not increase from the initial viable cell count cultured on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to have no replicative ability and are approved for use in the TIL expansion procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 3000 IU / mL of IL - 2. In some embodiments, the PBMCs are cultured in the presence of 60 ng / mL of OKT3 antibody and 6000 IU / mL of IL - 2. In some embodiments, the PBMCs are cultured in the presence of 60 ng / mL of OKT3 antibody and 3000 IU / mL of IL - 2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL - 2.

[0616] In some embodiments, if the total viable cell count cultured in the presence of OKT3 and IL-2 on the 7th and 14th days does not increase from the initial viable cell count cultured on day 0 of REP and / or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMC is considered to have no replication ability and is approved for use in the TIL expansion procedure described herein. In some embodiments, the PBMC is cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 1000 - 6000 IU / mL of IL-2. In some embodiments, the PBMC is cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 2000 - 5000 IU / mL of IL-2. In some embodiments, the PBMC is cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 2000 - 4000 IU / mL of IL- 2. In some embodiments, the PBMC is cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 2500 - 3500 IU / mL of IL-2. In some embodiments, the PBMC is cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0617] In some embodiments, the antigen-presenting feeder cells are PBMC. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TIL to antigen-presenting feeder cells in the second expansion is about 1:10, 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 TIL to antigen-presenting feeder cells in the second expansion is 1:50 - 1:300. In one embodiment, the ratio of TIL to antigen-presenting feeder cells in the second expansion is 1:100 - 1:200.

[0618] In one embodiment, the second rapid expansion procedure described herein is about 5×10 8 feeder cells: about 100×106 Requires a ratio of TIL. In certain embodiments, the second rapid expansion procedure described herein is about 7.5×10 8 Feeder cells: about 100×10 6 Requires a ratio of TIL. In another embodiment, the second rapid expansion procedure described herein is about 5×10 8 Feeder cells: about 50×10 6 Requires a ratio of TIL. In another embodiment, the second rapid expansion procedure described herein is about 7.5×10 8 Feeder cells: about 50×10 6 Requires a ratio of TIL. In yet another embodiment, the second expansion procedure described herein is about 5×10 8 Feeder cells: about 25×10 6 Requires TIL. In yet another embodiment, the second expansion procedure described herein is about 7.5×10 8 Feeder cells: about 25×10 6 Requires TIL. In yet another embodiment, the second rapid expansion requires twice the number of feeder cells of the second rapid expansion. In yet another embodiment, if the first priming expansion described herein requires about 2.5×10 8 Feeder cells, the second rapid expansion requires about 5×10 8 Feeder cells. In yet another embodiment, if the first priming expansion described herein requires about 2.5×10 8 Feeder cells, the second rapid expansion requires about 7.5×10 8 Feeder cells. In yet another embodiment, the second rapid expansion requires two times (2.0 times (2.0X)), 2.5 times, 3.0 times, 3.5 times, or 4.0 times the number of feeder cells of the first priming expansion.

[0619] In certain embodiments, the second rapid expansion procedure described herein requires an excess of feeder cells during the second rapid expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard unit of whole blood from a healthy allogeneic blood donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In certain embodiments, artificial antigen-prese...

Claims

1. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from said subject into a plurality of tumor fragments; (b) performing a first priming-expansion by culturing said first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen presenting cells (APCs) to produce a second TIL population, wherein said first priming-expansion is performed in a container comprising a first gas permeable surface area, and said first priming-expansion is performed for a first period of time of about 1-7 / 8 days to obtain said second TIL population, wherein said second TIL population is more numerous than said first TIL population; (c) performing a second rapid expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added to the second rapid expansion is at least twice the number of APCs added in step (b), and the second rapid expansion is performed for a second period of about 1-11 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second rapid expansion is performed in a container comprising a second gas permeable surface area. (d) harvesting the therapeutic TIL population obtained from step (c); (e) transferring the harvested TIL population from step (d) to an infusion bag.

2. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from said subject into a plurality of tumor fragments; (b) performing a first priming expansion by culturing the first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 1-7 / 8 days to obtain the second TIL population, the second TIL population being more numerous than the first TIL population; (c) performing a second rapid expansion by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 1-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (d) harvesting the therapeutic TIL population obtained from step (c).

3. 3. The method of claim 2, wherein in step (b), the cell culture medium further comprises antigen presenting cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

4. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first priming-expansion by culturing a first TIL population obtainable by processing a tumor sample from a tumor excised from a subject into a plurality of tumor fragments in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen presenting cells (APCs) to produce a second TIL population, wherein said first priming-expansion is performed in a container comprising a first gas permeable surface area, and said first priming-expansion is performed for a first period of about 1-7 / 8 days to obtain said second TIL population, and said second TIL population is cultured. producing a second TIL population, the L population being greater in number than the first TIL population; (b) performing a second rapid expansion by contacting the second TIL population with cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs in the second rapid expansion is at least twice the number of APCs in step (a), and the second rapid expansion is performed for a second period of about 1-11 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second rapid expansion is performed in a container comprising a second gas permeable surface area. (c) harvesting the therapeutic TIL population obtained from step (b).

5. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first priming-expansion by culturing a first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen presenting cells (APCs) to produce a second TIL population, said first priming-expansion being performed for a first period of time of about 1-7 / 8 days to obtain said second TIL population, said second TIL population being more numerous than said first TIL population; (b) performing a second rapid expansion by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of time of about 1-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (c) harvesting the therapeutic TIL population obtained from step (b).

6. 6. The method of claim 5, wherein in step (a), the cell culture medium further comprises antigen presenting cells (APCs), and the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

7. 7. The method of claim 1, wherein the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion ranges from about 1.5:1 to about 20:

1.

8. The method of claim 7, wherein the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion ranges from about 1.5:1 to 10:

1.

9. The method of claim 1, 3 or 6, wherein the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion ranges from about 2:1 to about 5:

1.

10. The method of claim 1, 3 or 6, wherein the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion ranges from about 2:1 to about 3:

1.

11. 10. The method of claim 1, wherein the ratio of the number of APCs in the second rapid expansion to the number of APCs in the first priming expansion is about 2:

1.

12. The number of APCs in the first priming expansion is about 1.0 x 10 ６ APC / cm ２ ~Approx. 4.5×10 ６ APC / cm ２ and the number of APCs in the second rapid expansion is in the range of about 2.5×10 ６ APC / cm ２ ~Approx. 7.5×10 ６ APC / cm ２ Within the scope of The method according to claim 1 or 3 or 6.

13. The number of APCs in the first priming expansion is about 1.5×10 ６ APC / cm ２ ~Approx. 3.5×10 ６ APC / cm ２ and the number of APCs in the second rapid expansion is in the range of about 3.5×10 ６ APC / cm ２ ~Approx. 6.0×10 ６ APC / cm ２ The method according to claim 1, 3 or 6, wherein the range is

14. The number of APCs in the first priming expansion is about 2.0 x 10 ６ APC / cm ２ ~Approx. 3.0×10 ６ APC / cm ２ and the number of APCs in the second rapid expansion is in the range of about 4.0 x 10 ６ APC / cm ２ ~Approx. 5.5×10 ６ APC / cm ２ The method according to claim 1, 3 or 6, wherein the range is

15. The number of APCs in the first priming expansion is about 1×10 ８ APC ~ approx. 3.5 × 10 ８ APCs, and the number of APCs in the second rapid expansion is about 3.5×10 ８ APC ~ approx. 1 x 10 ９ The method of claim 1, 3 or 6, which is in the range of APC.

16. The number of APCs in the first priming expansion is about 1.5×10 ８ APC ~ approx. 3 x 10 ８ APCs, and the number of APCs in the second rapid expansion is about 4×10 ８ APC ~ approx. 7.5 × 10 ８ The method of claim 1, 3 or 6, which is in the range of APC.

17. The number of APCs in the first priming expansion is about 2×10 ８ APC ~ approx. 2.5 × 10 ８ APCs, and the number of APCs in the second rapid expansion is about 4.5×10 ８ APC ~ approx. 5.5 × 10 ８ The method of claim 1, 3 or 6, which is in the range of APC.

18. Approximately 2.5 x 10 ８ APCs were added to the first priming expansion, and 5×10 ８ The method of claim 1, 3 or 6, wherein APCs are added to the second rapid expansion.

19. The method of any of claims 1 to 18, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is from about 1.5:1 to about 100:

1.

20. The method of any of claims 1 to 18, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 50:

1.

21. The method of any of claims 1 to 18, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 25:

1.

22. The method of any of claims 1 to 15, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 20:

1.

23. The method of any of claims 1 to 15, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 10:

1.

24. The method of any of claims 1 to 18, wherein the second TIL population is at least 50-fold more numerous than the first TIL population.

25. The method further comprises, after the step of harvesting the therapeutic TIL population, The method of any of claims 2 to 6, comprising performing the additional step of transferring the harvested therapeutic TIL population to an infusion bag.

26. The method of any of claims 2 to 25, wherein the plurality of tumor fragments are distributed into a plurality of separate containers, in each of which the second TIL population is obtained from the first TIL population in the first priming expansion step, the third TIL population is obtained from the second TIL population in the second rapid expansion step, and the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to produce the harvested TIL population.

27. 27. The method of claim 26, wherein the plurality of separate containers comprises at least two separate containers.

28. 27. The method of claim 26, wherein the plurality of separate containers comprises between 2 and 20 separate containers.

29. 27. The method of claim 26, wherein the plurality of separate containers comprises between 2 and 10 separate containers.

30. 27. The method of claim 26, wherein the plurality of separate containers comprises 2 to 5 separate containers.

31. The method of any of claims 26-30, wherein each of the separate containers comprises a first gas permeable surface area.

32. The method of any of claims 2 to 25, wherein the multiple tumor fragments are distributed in a single container.

33. 33. The method of claim 32, wherein the single container comprises a first gas permeable surface area.

34. 34. The method of claim 31 or 33, wherein in the first priming expansion step, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered onto the first gas permeable surface area at an average thickness of about 1 cell layer to about 3 cell layers.

35. 34. The method of claim 33, wherein in the first priming expansion step, the APCs are layered onto the first gas permeable surface area at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

36. 34. The method of claim 33, wherein in the first priming expansion step, the APCs are layered onto the first gas permeable surface area at an average thickness of about two cell layers.

37. 37. The method of any of claims 34-36, wherein in the second rapid expansion step, the APCs are layered onto the first gas permeable surface area at a thickness of about 3 cell layers to about 5 cell layers.

38. 38. The method of claim 37, wherein in the second rapid expansion step, the APCs are layered onto the first gas permeable surface area at a thickness of about 3.5 cell layers to about 4.5 cell layers.

39. 40. The method of claim 38, wherein in the second rapid expansion step, the APCs are layered onto the first gas permeable surface area at a thickness of about four cell layers.

40. 26. The method of any of claims 2 to 25, wherein in the first priming expansion step, the first priming expansion is performed in a first container comprising a first gas permeable surface area, and in the second rapid expansion step, the second rapid expansion is performed in a second container comprising a second gas permeable surface area.

41. 41. The method of claim 40, wherein the second container is larger than the first container.

42. 42. The method of claim 40 or 41, wherein in the first priming expansion step, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered onto the first gas permeable surface area at an average thickness of about 1 cell layer to about 3 cell layers.

43. 42. The method of claim 41, wherein in the first priming expansion step, the APCs are layered onto the first gas permeable surface area at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

44. 44. The method of claim 43, wherein in the first priming expansion step, the APCs are layered onto the first gas permeable surface area at an average thickness of about two cell layers.

45. 45. The method of any of claims 40-44, wherein in the second rapid expansion step, the APCs are layered onto the second gas permeable surface area at an average thickness of about 3 cell layers to about 5 cell layers.

46. 46. ​​The method of claim 45, wherein in the second rapid expansion step, the APCs are layered onto the second gas permeable surface area at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

47. 46. ​​The method of claim 45, wherein in the second rapid expansion step, the APCs are layered onto the second gas permeable surface area at an average thickness of about four cell layers.

48. 40. The method of any of claims 2 to 39, wherein for each vessel in which the first priming expansion is performed on a first TIL population, the second rapid expansion is performed in the same vessel on a second TIL population produced from such first TIL population.

49. 49. The method of claim 48, wherein each container comprises a first gas permeable surface area.

50. 50. The method of claim 49, wherein in the first priming expansion step, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered onto the first gas permeable surface area at an average thickness of about 1 cell layer to about 3 cell layers.

51. 51. The method of claim 50, wherein in said first priming expansion step, said APCs are layered onto said first gas permeable surface area at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

52. 52. The method of claim 51, wherein in the first priming expansion step, the APCs are layered onto the first gas permeable surface area at an average thickness of about two cell layers.

53. 53. The method of any of claims 49-52, wherein in the second rapid expansion step, the APCs are layered onto the first gas permeable surface area at an average thickness of about 3 cell layers to about 5 cell layers.

54. 54. The method of claim 53, wherein in said second rapid expansion step, said APCs are layered onto said first gas permeable surface area at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

55. 55. The method of claim 54, wherein in the second rapid expansion step, the APCs are layered onto the first gas permeable surface area at an average thickness of about four cell layers.

56. 50. The method of any of claims 2-32, 40, 41, and 48, wherein for each container in which the first priming-expansion is performed on a first TIL population in the first priming-expansion step, the first container comprises a first surface area, the cell culture medium comprises antigen presenting cells (APCs), the APCs are layered on the first gas permeable surface area, and a ratio of an average number of layers of APCs layered in the first priming-expansion step to an average number of layers of APCs layered in the second rapid-expansion step ranges from about 1:1.1 to about 1:

10.

57. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.2 to about 1:

8.

58. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.3 to about 1:

7.

59. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.4 to about 1:

6.

60. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.5 to about 1:

5.

61. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.6 to about 1:

4.

62. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.7 to about 1:3.

5.

63. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.8 to about 1:

3.

64. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step ranges from about 1:1.9 to about 1:2.

5.

65. 57. The method of claim 56, wherein the ratio of the average number of layers of APCs layered in the first priming expansion step to the average number of layers of APCs layered in the second rapid expansion step is about 1:

2.

66. 66. The method of any of claims 1-65, wherein after the second rapid expansion step for 2-3 days, the cell culture medium is supplemented with additional IL-2.

67. 67. The method of any of claims 1-66, further comprising, in the step of harvesting the therapeutic TIL population, cryopreserving the harvested TIL population using a cryopreservation process.

68. 30. The method of claim 1 or 25, further comprising the step of cryopreserving the infusion bag.

69. The cryopreservation process comprises a 1:1 ratio of harvested TIL population and cryopreservation.

69. The method of claim 67 or 68, which is carried out using a storage medium.

70. The method of any one of claims 1 to 69, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

71. 71. The method of claim 70, wherein the PBMCs are irradiated and allogeneic.

72. In the first priming expansion step, the cell culture medium includes peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the first priming expansion step is about 2.5×10 ８ The method according to any one of claims 1 to 71, wherein

73. In the second rapid expansion step, the antigen presenting cells (APCs) in the cell culture medium are peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the second rapid expansion step is about 5×10 ８ The method according to any one of claims 1 to 72, wherein

74. The method of any one of claims 1 to 66, wherein the antigen-presenting cell is an artificial antigen-presenting cell.

75. 75. The method of any of claims 1-74, wherein the harvesting of the therapeutic TIL population is performed using a membrane-based cell processing system.

76. The method of any of claims 1-75, wherein said harvesting in the step of harvesting said therapeutic TIL population is performed using a LOVO cell processing system.

77. The plurality of fragments includes about 60 fragments per container in a first priming expansion step, each fragment being about 27 mm ３ The method of any one of claims 1 to 76, having a volume of

78. The plurality of pieces includes about 30 to about 60 pieces and has a total volume of about 1300 mm ３ ~Approx. 1500mm ３ The method according to any one of claims 1 to 77, wherein

79. The plurality of pieces includes about 50 pieces and has a total volume of about 1350 mm ３ 79. The method of claim 78, wherein:

80. 80. The method of any of claims 1-79, wherein the plurality of fragments comprises about 50 fragments and has a total mass of about 1 gram to about 1.5 grams.

81. 81. The method of any of claims 1-80, wherein the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

82. 82. The method of any of claims 1-81, wherein the IL-2 concentration is from about 10,000 IU / mL to about 5,000 IU / mL.

83. 83. The method of any of claims 1-82, wherein the IL-2 concentration is about 6,000 IU / mL.

84. 26. The method of claim 1 or 25, wherein the infusion bag in the step of transferring the harvested therapeutic TIL population to an infusion bag is an infusion bag containing HypoThermosol.

85. 70. The method of any of claims 67 to 69, wherein the cryopreservation medium comprises dimethylsulfoxide (DMSO).

86. 86. The method of claim 85, wherein the cryopreservation medium comprises 7% to 10% DMSO.

87. 87. The method of any of claims 1-86, wherein the first period of the first priming expansion step and the second period of the second rapid expansion step are each performed individually within a period of 5 days, 6 days, or 7 days.

88. 87. The method of any of claims 1-86, wherein the first period of the first priming extension step is performed within a period of 5, 6, or 7 days.

89. 87. The method of any of claims 1-86, wherein the second period of the second rapid expansion step is performed within a period of 7 days, 8 days, or 9 days.

90. 87. The method of any of claims 1-86, wherein the first period of the first priming expansion step and the second period of the second rapid expansion step are each performed individually within a period of 7 days.

91. 87. The method of any of claims 1-86, wherein the first priming-expansion step through the harvesting of the therapeutic TIL population is performed within a period of about 14 to about 16 days.

92. The method of any of claims 1-86, wherein the first priming-expansion step through the harvesting of the therapeutic TIL population is performed within a period of about 15 to about 16 days.

93. The method of any of claims 1 to 86, wherein the first priming-expansion step through the harvesting of the therapeutic TIL population is performed within a period of about 14 days.

94. The method of any of claims 1 to 86, wherein the first priming-expansion step through the harvesting of the therapeutic TIL population is performed within a period of about 15 days.

95. The method of any of claims 1 to 86, wherein the first priming-expansion step through the harvesting of the therapeutic TIL population is performed within a period of about 16 days.

96. The method of any of claims 1 to 86, further comprising the step of cryopreserving the harvested therapeutic TIL population using a cryopreservation process, wherein the first priming-expansion step through the harvesting of the therapeutic TIL population and the cryopreservation step are performed within 16 days or less.

97. The therapeutic TIL population collected in the step of collecting the therapeutic TIL population is TIL 94. The method of any one of claims 1 to 93, comprising a sufficient therapeutically effective dose of said TILs.

98. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3×10 １０ ~Approx. 13.7×10 １０ 98. The method of claim 97, wherein:

99. The method of any one of claims 1 to 98, wherein the third TIL population in the second rapid expansion step provides increased efficacy, increased interferon gamma production, and / or increased polyclonality.

100. 99. The method of any one of claims 1 to 98, wherein the third population of TILs in the second rapid expansion step provides at least 1-5-fold more interferon gamma production compared to TILs prepared by an 18 day or longer process.

101. 99. The method of any one of claims 1 to 98, wherein effector T cells and / or central memory T cells obtained from the third TIL population in the second rapid expansion step exhibit increased CD8 and CD28 expression relative to effector T cells and / or central memory T cells obtained from the second TIL population in the first priming expansion step.

102. The method of any one of claims 1 to 101, wherein the therapeutic TIL population from the step of harvesting the therapeutic TIL population is infused into a patient.

103. 1. A method for treating a subject having cancer, comprising: (a) obtaining and / or receiving a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from said subject into a plurality of tumor fragments; (b) performing a first priming-expansion by culturing said first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen presenting cells (APCs) to produce a second TIL population, said first priming-expansion being performed in a vessel comprising a first gas permeable surface area, said first priming-expansion being performed for about 1-7 / 8 days to obtain said second TIL population, said second TIL population being at least 50-fold greater in number than said first TIL population; (c) performing a second rapid expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added to the second rapid expansion is at least twice the number of APCs added in step (b), and the second rapid expansion is performed for about 1-11 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second rapid expansion is performed in a vessel comprising a second gas permeable surface area. (d) harvesting the therapeutic TIL population obtained from step (c); (e) transferring the harvested TIL population from step (d) into an infusion bag; (f) administering a therapeutically effective dose of the TIL population from step (e) to the subject.

104. A sufficient number of TILs to administer a therapeutically effective dose in step (f) is about 2.3×10 １０ ~Approx. 13.7×10 １０ The method of claim 103, wherein:

105. The method of claim 103, wherein the antigen presenting cell (APC) is a PBMC.

106. Prior to administering a therapeutically effective dose of TIL cells in step (f), non-myeloablative The method of any of claims 103 to 105, wherein the patient has been administered a targeted lymphodepleting regimen.

107. The non-myeloablative lymphodepleting regimen comprises cyclophosphamide at 60 mg / m ２ / day for 2 days, followed by fludarabine at 25 mg / m ２ 107. The method of claim 106, comprising administering at a dose of 100 mg / day for 5 days.

108. The method of any of claims 103-107, further comprising treating said patient with a high dose IL-2 regimen beginning the day after administration of said TIL cells to said patient in step (f).

109. 109. The method of claim 108, wherein the high dose IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15 minute bolus intravenous infusion every 8 hours until tolerated.

110. The method of any one of claims 103 to 109, wherein the third TIL population in step (b) provides increased efficacy, increased interferon gamma production, and / or increased polyclonality.

111. 110. The method of any one of claims 103-109, wherein the third population of TILs in step (c) provides at least 1-fold to 5-fold more interferon gamma production compared to TILs prepared by a process of 16 days or more.

112. 110. The method of any one of claims 103 to 109, wherein effector T cells and / or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression relative to effector T cells and / or central memory T cells obtained from the second cell population in step (b).

113. The method of any one of claims 103 to 112, wherein the cancer is a solid tumor.

114. 113. The method of any one of claims 103-112, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

115. 115. The method of claim 114, wherein the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

116. 116. The method of claim 115, wherein the cancer is melanoma.

117. 116. The method of claim 115, wherein the cancer is HNSCC.

118. 116. The method of claim 115, wherein the cancer is cervical cancer.

119. 116. The method of claim 115, wherein the cancer is NSCLC.

120. 116. The method of claim 115, wherein the cancer is glioblastoma (including GBM).

121. 116. The method of claim 115, wherein the cancer is a gastrointestinal cancer.

122. The method of any one of claims 103 to 121, wherein the cancer is a hypermutated cancer.

123. The method of any one of claims 103 to 121, wherein the cancer is a childhood hypermutated cancer.

124. The method of any one of claims 103 to 123, wherein the container is a sealed container.

125. The method of any one of claims 103 to 124, wherein the vessel is a G vessel.

126. The method of any one of claims 103 to 125, wherein the container is GREX-10.

127. The method of any one of claims 103 to 125, wherein the sealed container comprises GREX-100.

128. The method of any one of claims 103 to 125, wherein the sealed container comprises GREX-500.

129. A therapeutic population of tumor infiltrating lymphocytes (TILs) produced by the method of any of claims 1-128.

130. A therapeutic population of tumor infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, said therapeutic TIL population providing increased efficacy, increased interferon gamma production, and / or increased polyclonality.

131. The therapeutic TIL population of claim 129 or claim 130, which provides increased interferon gamma production.

132. The therapeutic TIL population of claim 129 or claim 130, which provides increased polyclonality.

133. The therapeutic TIL population of claim 129 or claim 130, which provides increased efficacy.

134. The therapeutic TIL population of any of claims 129-133, wherein the therapeutic TIL population is capable of at least 1-fold greater interferon gamma production compared to TILs prepared by a process of 16 days or longer.

135. The therapeutic TIL population of any of claims 129-133, wherein said therapeutic TIL population is capable of at least 2-fold greater interferon gamma production compared to TILs prepared by a process of 16 days or longer.

136. The therapeutic TIL population of any of claims 129-133, wherein said therapeutic TIL population is capable of at least 3-fold greater interferon gamma production compared to TILs prepared by a process of 16 days or longer.

137. A therapeutic tumor infiltrating lymphocyte (TIL) population, wherein the therapeutic TIL population is capable of at least 1-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any antigen presenting cells (APCs).

138. The therapeutic TIL population of claim 137, wherein the therapeutic TIL population is capable of at least 2-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any APCs.

139. The therapeutic TIL population of claim 138, wherein the therapeutic TIL population is capable of at least three times more interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any APCs.

140. A therapeutic tumor infiltrating lymphocyte (TIL) population, wherein the therapeutic TIL population is capable of at least 1-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any OKT3.

141. The therapeutic TIL population of claim 140, wherein the therapeutic TIL population is capable of at least 2-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any OKT3.

142. The therapeutic TIL population of claim 140, wherein the therapeutic TIL population is capable of at least 3-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any OKT3.

143. The therapeutic TIL population is capable of at least 1-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any antigen presenting cells (APCs) or any OKT3.

144. The therapeutic TIL population of claim 143, wherein the therapeutic TIL population is capable of at least 2-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any antigen presenting cells (APCs) or any OKT3.

145. The therapeutic TIL population of claim 143, wherein the therapeutic TIL population is capable of at least 3-fold greater interferon gamma production compared to TILs prepared by a process in which the first TIL expansion is performed without the addition of any antigen presenting cells (APCs) or any OKT3.

146. 146. A tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population according to any one of claims 126-145 and a pharma- ceutically acceptable carrier.

147. A sterile infusion bag comprising the TIL composition of claim 143.

148. 143. A cryopreserved preparation of a therapeutic TIL population according to any of claims 129-142.

149. Therapeutic TIL populations and cryopreservations according to any of claims 129 to 145. A tumor infiltrating lymphocyte (TIL) composition comprising a storage medium.

150. The TIL composition of claim 149, wherein the cryopreservation medium comprises DMSO.

151. The TIL composition of claim 150, wherein the cryopreservation medium comprises 7-10% DMSO.

152. 152. A cryopreserved preparation of the TIL composition of any of claims 146-151.

153. A tumor infiltrating lymphocyte (TIL) composition according to any of claims 146 to 152 for use as a medicament.

154. A tumor infiltrating lymphocyte (TIL) composition according to any of claims 146 to 152 for use in the treatment of cancer.

155. A tumor infiltrating lymphocyte (TIL) composition according to any of claims 146 to 152 for use in the treatment of solid tumor cancer.

156. 153. The tumor infiltrating lymphocyte (TIL) composition of any of claims 146-152 for use in the treatment of a cancer selected from melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

157. 153. The tumor infiltrating lymphocyte (TIL) composition of any of claims 146-152 for use in the treatment of a cancer selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

158. The TIL composition of any of claims 146 to 152 for use in the treatment of cancer, wherein the cancer is melanoma.

159. The TIL composition of any of claims 146 to 152 for use in the treatment of cancer, wherein the cancer is HNSCC.

160. A TIL composition according to any of claims 146 to 152 for use in the treatment of a cancer which is cervical cancer.

161. The TIL composition of any of claims 146 to 152 for use in treating cancer, wherein said cancer is NSCLC.

162. A TIL composition according to any of claims 146 to 152 for use in the treatment of cancer, wherein the cancer is glioblastoma (including GBM).

163. The TIL composition of any of claims 146 to 152 for use in the treatment of cancer, wherein the cancer is a gastrointestinal cancer.

164. A TIL composition according to any of claims 146 to 152 for use in the treatment of cancer, wherein said cancer is a hypermutated cancer.

165. The TIL composition of any of claims 146 to 152 for use in treating cancer, wherein said cancer is a pediatric hypermutated cancer.

166. Use of a tumor infiltrating lymphocyte (TIL) composition according to any of claims 146 to 152 in a method for treating cancer in a subject comprising administering to the subject a therapeutically effective dose of the TIL composition.

167. The use of the TIL composition of claim 166, wherein the cancer is a solid tumor.

168. The use of the TIL composition of claim 166, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

169. The use of the TIL composition of claim 166, wherein the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

170. The use of the TIL composition of claim 166, wherein the cancer is melanoma.

171. The use of the TIL composition of claim 166, wherein the cancer is HNSCC.

172. The use of the TIL composition of claim 166, wherein the cancer is cervical cancer.

173. The use of the TIL composition of claim 166, wherein the cancer is NSCLC.

174. The use of the TIL composition of claim 166, wherein the cancer is glioblastoma (including GBM).

175. The use of the TIL composition of claim 166, wherein the cancer is a gastrointestinal cancer.

176. The use of the TIL composition of claim 166, wherein the cancer is a hypermutated cancer.

177. The use of the TIL composition of claim 166, wherein the cancer is a pediatric hypermutated cancer.

178. A tumor infiltrating lymphocyte (TIL) composition according to any of claims 143 to 152 for use in a method of treating cancer in a subject comprising administering to the subject a therapeutically effective dose of the TIL composition.

179. The TIL composition of claim 178, wherein the cancer is a solid tumor.

180. The TIL composition of claim 178, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

181. The TIL composition of claim 178, wherein the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

182. A method of treating cancer in a subject comprising administering to the subject a therapeutically effective dose of a tumor infiltrating lymphocyte (TIL) composition according to any one of claims 143 to 152.

183. 183. The method of claim 182, wherein the cancer is a solid tumor.

184. The cancer may be melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

183. The method of claim 182, wherein the cancer is selected from the group consisting of:

185. 183. The method of claim 182, wherein the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

186. 183. The method of claim 182, wherein the cancer is melanoma.

187. 183. The method of claim 182, wherein the cancer is HNSCC.

188. 183. The method of claim 182, wherein the cancer is cervical cancer.

189. 183. The method of claim 182, wherein the cancer is NSCLC.

190. 183. The method of claim 182, wherein the cancer is glioblastoma (including GBM).

191. 183. The method of claim 182, wherein the cancer is a gastrointestinal cancer.

192. The method of claim 182, wherein the cancer is a hypermutated cancer.

193. The method of claim 182, wherein the cancer is a pediatric hypermutated cancer.

194. 1. A method for expanding T cells, comprising: (a) performing a first priming expansion of a first T cell population obtained from a donor by culturing said first T cell population to result in expansion and prime activation of said first T cell population; (b) after the activation of the first T cell population primed in step (a) begins to decay, performing a second rapid expansion of the first T cell population by culturing the first T cell population to effect growth and promote the activation of the first T cell population to obtain a second T cell population; (c) harvesting the second population of T cells.

195. 195. The method of claim 194, wherein the first priming extension of step (a) is performed for a period of up to 7 days.

196. 196. The method of claim 194 or 195, wherein the second rapid expansion of step (b) is performed for a period of up to 11 days.

197. 200. The method of claim 196, wherein the second rapid expansion of step (b) is performed for a period of up to 9 days.

198. 200. The method of any of claims 194-197, wherein the first priming extension of step (a) is performed during a period of 7 days and the second rapid expansion of step (b) is performed during a period of 9 days.

199. 195. The method of claim 194, wherein the first priming extension of step (a) is performed for a period of up to 8 days.

200. 196. The method of claim 194 or 195, wherein the second rapid expansion of step (b) is performed for a period of up to 8 days.

201. 200. The method of any of claims 194-197, wherein the first priming expansion of step (a) is performed during a period of 8 days and the second rapid expansion of step (b) is performed during a period of 8 days.

202. The method of any of claims 194-201, wherein in step (a), the first population of T cells is cultured in a first culture medium comprising OKT-3 and IL-2.

203. 203. The method of claim 202, wherein the first culture medium comprises OKT-3, IL-2, and antigen presenting cells (APCs).

204. The method of any of claims 194-201, wherein in step (b), the first population of T cells is cultured in a second culture medium comprising OKT-3, IL-2, and antigen presenting cells (APCs).

205. 202. The method of any of claims 194-201, wherein in step (a), the first T cell population is cultured in a first culture medium in a vessel comprising a first gas permeable surface, the first culture medium optionally comprising OKT-3, IL-2, and optionally a first antigen presenting cell (APC) population, the first APC population being exogenous to a donor of the first T cell population, the first APC population being layered onto the first gas permeable surface, and wherein in step (b), the first T cell population is cultured in a second culture medium in the vessel, the second culture medium comprising OKT-3, IL-2, and a second APC population, the second APC population being exogenous to a donor of the first T cell population, the second APC population being layered onto the first gas permeable surface, the second APC population being larger than the first APC population.

206. The method of claim 205, wherein the ratio of the number of APCs in the second APC population to the number of APCs in the first APC population is about 2:

1.

207. The number of APCs in the first population of APCs is about 2.5×10 ８ and the number of APCs in the second population of APCs is about 5×10 ８ The method of claim 205 or 206,

208. The method of any of claims 205-207, wherein in step (a), the first population of APCs is layered onto the first gas permeable surface at an average thickness of two layers of APCs.

209. The method of any of claims 205-208, wherein in step (b), the second population of APCs is layered onto the first gas permeable surface at an average thickness in the range of 4 to 8 layers of APCs.

210. 210. The method of any of claims 205-209, wherein the ratio of the average number of layers of APC layered onto the first gas permeable surface in step (b) to the average number of layers of APC layered onto the first gas permeable surface in step (a) is 2:

1.

211. The method of any of claims 205 to 210, wherein the APC is a peripheral blood mononuclear cell (PBMC).

212. The method of any of claims 205-211, wherein the APCs comprise PBMCs that are irradiated and exogenous to the donor of said first T cell population.

213. The method of any of claims 202-209, wherein the T cells are tumor infiltrating lymphocytes (TILs).

214. The method of any of claims 202-209, wherein the T cells are bone marrow infiltrating lymphocytes (MIL).

215. The method of any of claims 202-209, wherein the T cells are peripheral blood lymphocytes (PBLs).

216. The method of any of claims 1 to 215, wherein the cell culture medium is a synthetic medium and / or a serum-free medium.

217. 217. The method of claim 216, wherein the synthetic medium comprises (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

218. The method of any of claims 216-217, wherein said serum-free or synthetic medium comprises a basal cell culture medium and a serum supplement and / or serum replacement.

219. The basal cell culture medium may be CTS(TM) OpTmizer(TM) T-cell Expansion Basal Medium, CTS(TM) OpTmizer(TM) T-Cell Expansion SFM, CTS(TM) AIM-V Medium, CTS(TM) AIM-V SFM, LymphoONE(TM) T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

220. 220. The method of any of claims 218-219, wherein the serum supplement or serum replacement is selected from CTS™ OpTmizer T-Cell Expansion Serum Supplement and CTS™ Immune Cell Serum Replacement.

221. 221. The method of any of claims 216-220, wherein the cell culture medium comprises one or more albumins or albumin substitutes.

222. 222. The method of any of claims 216-221, wherein the cell culture medium comprises one or more amino acids.

223. 223. The method of any of claims 216-222, wherein the cell culture medium comprises one or more vitamins, one or more transferrin or transferrin substitutes.

224. 224. The method of any of claims 216-223, wherein the cell culture medium comprises one or more antioxidants, one or more insulin or insulin substitutes.

225. 225. The method of any of claims 216-224, wherein the cell culture medium comprises one or more collagen precursors, one or more antibiotics, and one or more trace elements.

226. 226. The method of any of claims 216-225, wherein the cell culture medium comprises albumin.

227. The cell culture medium contains albumin, glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element moiety Ag ＋ , Al ３＋ , Ba ２＋ , Cd ２＋ , Co ２＋ , Cr ３＋ , Ge ４＋ , Se ４＋ , Br, T, Mn ２＋ , P, Si ４＋ , V ５＋ , Mo ６＋ , Ni ２＋ , Rb ＋ , Sn ２＋ , and Zr ４＋ and one or more components selected from the group consisting of compounds containing

228. 228. The method of any of claims 216-227, wherein the cell culture medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

229. 229. The method of any of claims 216-228, wherein the cell culture medium has a total serum replacement concentration (volume %) from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the cell culture medium.

230. 230. The method of any of claims 216-229, wherein the cell culture medium has a total serum replacement concentration of about 3%, about 5%, or about 10% of the total volume of the cell culture medium.

231. 231. The method of any of claims 216-230, wherein the cell culture medium further comprises glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM.

232. 232. The method of any of claims 216-231, wherein the cell culture medium further comprises glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

233. 233. The method of any of claims 216-232, wherein the cell culture medium further comprises 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM.

234. 234. The method of any of claims 216-233, wherein the cell culture medium further comprises 2-mercaptoethanol at a concentration of about 55 mM.

235. 235. The method of any of claims 216-234, wherein the cell culture medium comprises a synthetic medium as described in International PCT Publication No. WO / 1998 / 030679.

236. The cell culture medium may comprise glycine in the range of about 5-200 mg / L, L-histidine in the range of about 5-250 mg / L, L-isoleucine in the range of about 5-300 mg / L, L-methionine in the range of about 5-200 mg / L, L-phenylalanine in the range of about 5-400 mg / L, L-proline in the range of about 1-1000 mg / L, L-hydroxyproline in the range of about 1-45 mg / L, L-serine in the range of about 1-250 mg / L, L-threonine in the range of about 10-500 mg / L, L-tryptophan in the range of about 2-110 mg / L, L-tyrosine in the range of about 3-175 mg / L, L-valine in the range of about 5-500 mg / L, thiamine in the range of about 1-20 mg / L, reduced glutathione in the range of about 1-20 mg / L, riboflavin ...

236. The method of any of claims 216-235, comprising L-ascorbic acid-2-phosphate in the range of up to 200 mg / L, iron-saturated transferrin in the range of about 1-50 mg / L, insulin in the range of about 1-100 mg / L, sodium selenite in the range of about 0.000001-0.0001 mg / L, and / or albumin (e.g., AlbuMAX® I) in the range of about 5000-50,000 mg / L.

237. 237. The method of any of claims 216-236, wherein the cell culture medium comprises one or more of the non-trace element moieties at a concentration range listed in the column under the heading "Concentration Range in 1x Medium" in Table A provided herein.

238. The method of any of claims 216 to 237, wherein the osmolarity of the cell culture medium is about 260 to 350 mOsmol.

239. The method of any of claims 216-238, wherein the cell culture medium further comprises about 3.7 g / L, or about 2.2 g / L, of sodium bicarbonate.

240. 240. The method of any of claims 216-239, wherein the cell culture medium further comprises L-glutamine (at a final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, at a final concentration of about 100 μM), and / or 2-mercaptoethanol (at a final concentration of about 100 μM).

241. 241. The method of any of claims 216-240, wherein the cell culture medium in the first and / or second gas permeable container is devoid of β-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

242. The cell culture medium is CTS OpTmizer T-Cell Expansion 241. The method of any of claims 216-240, comprising SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

243. 241. The method of any of claims 216-240, wherein said cell culture medium comprises CTS™ OpTmizer™ T-Cell Expansion Basal Medium supplemented with CTS™ OpTmizer™ T-Cell Expansion Supplement (26mL / L), and 3% CTS™ Immune Cell SR, and 2mM Glutamax, and optionally further comprising 6,000 IU / mL of IL-2.

244. 241. The method of any of claims 216-240, wherein the cell culture medium comprises CTS™ OpTmizer™ T-Cell Expansion Supplement (26mL / L), and CTS™ OpTmizer™ T-Cell Expansion Basal Medium supplemented with 3% CTS™ Immune Cell SR, 2mM Glutamax, and optionally further comprising 3,000 IU / mL IL-2.

245. The method of any of claims 1-244, wherein the tumor sample is one or more mini biopsies, core biopsies, or needle biopsies of the tumor in the subject.

246. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (i) isolating a first population of TILs from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor in a subject, said tumor sample in a first cell culture medium comprising IL-2. and / or receiving the cells by culturing the cells at 4° C. for about 3 days. (ii) performing a first priming-expansion by culturing the first TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second TIL population, wherein the first priming-expansion is performed in a container comprising a first gas permeable surface area, and wherein the first priming-expansion is performed for a first period of about 7 or 8 days to obtain the second TIL population, wherein the second TIL population is more numerous than the first TIL population; (iii) performing a second rapid expansion by supplementing the second cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added to the second rapid expansion is at least twice the number of APCs added in step (ii), and the second rapid expansion is performed for a second period of about 11 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second rapid expansion is performed in a container comprising a second gas permeable surface area. (iv) harvesting the therapeutic TIL population obtained from step (iii); (v) transferring the harvested TIL population from step (iv) to an infusion bag.

247. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (i) obtaining and / or receiving a first population of TILs from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor in a subject by culturing said tumor sample in a first cell culture medium comprising IL-2 for about 3 days; (ii) performing a first priming expansion by culturing the first TIL population in a second cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed for a first period of about 7 or 8 days to obtain the second TIL population, the second TIL population being more numerous than the first TIL population; (iii) performing a second rapid expansion by contacting the second TIL population with a third cell culture medium comprising IL-2, OKT-3, and APC to produce a third TIL population, wherein the second rapid expansion is performed for a second period of about 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; (iv) harvesting the therapeutic TIL population obtained from step (iii).

248. The method of claim 246 or 247, wherein after day 5 of the second period, the culture is divided into two or more subcultures, each subculture being supplemented with an additional amount of the third culture medium and cultured for about 6 days.

249. 249. The method of claim 248, wherein after day 5 of the second period of time, the culture is split into up to five subcultures.

250. 250. The method of any of claims 246-249, wherein all steps of the method are completed in about 22 days.

251. 1. A method for expanding T cells, comprising: (i) a first priming expansion of a first T cell population from a tumor sample obtained from one or more mini-biopsies, core biopsies, or needle biopsies of a tumor in a donor by culturing said first T cell population to result in growth and prime activation of said first T cell population; (ii) after the activation of the first T cell population primed in step (a) begins to decay, performing a second rapid expansion of the first T cell population by culturing the first T cell population to result in growth and promote the activation of the first T cell population to obtain a second T cell population; (iv) harvesting the second population of T cells.

252. The method of any of claims 245-251, wherein the tumor sample is obtained from multiple core biopsies.

253. 253. The method of claim 252, wherein said multiple core biopsies are selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, and 10 core biopsies.

254. 1. A tumor infiltrating lymphocyte (TIL) or expanded tumor infiltrating lymphocyte (TIL) composition comprising: i) a population of tumor infiltrating lymphocytes (TILs); ii) a synthetic or serum-free medium optionally comprising (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin,

255. The TIL or expanded TIL composition of claim 245, wherein the synthetic or serum-free medium comprises (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

256. The TIL or expanded TIL composition of claim 245 or 246, wherein the synthetic or serum-free medium comprises a basal cell medium and a serum supplement and / or serum replacement.

257. The basal cell culture medium includes CTS(TM) OpTmizer(TM) T-cell Expansion Basal Medium, CTS(TM) OpTmizer(TM) T-Cell Expansion SFM, CTS(TM) AIM-V Medium, CTS(TM) AIM-V SFM, LymphoONE(TM) T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 248. The TIL or expanded TIL composition of claim 247, including, but not limited to, 1640, F-10, F-12, Minimum Essential Medium (alpha MEM), Glasgow Minimum Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

258. 258. The TILs or expanded TILs of any of claims 256-257, wherein the serum supplement or serum replacement is selected from the group consisting of CTS™ OpTmizer T-Cell Expansion Serum Supplement and CTS™ Immune Cell Serum Replacement.

259. 259. The TIL or expanded TIL composition of any of claims 254-258, wherein said synthetic or serum-free medium comprises one or more albumins or albumin substitutes.

260. 260. The TIL or expanded TIL composition of any of claims 254-259, wherein said synthetic or serum-free medium comprises one or more amino acids.

261. 261. The TIL or expanded TIL composition of any of claims 254-260, wherein the synthetic or serum-free medium comprises one or more vitamins, one or more transferrin or transferrin substitutes.

262. 262. The TIL or expanded TIL composition of any of claims 254-261, wherein the synthetic or serum-free medium comprises one or more antioxidants, one or more insulin or insulin substitutes.

263. The TIL or expanded TIL composition of any of claims 254-262, wherein the synthetic or serum-free medium comprises one or more collagen precursors, one or more antibiotics, and one or more trace elements.

264. 264. The TIL or expanded TIL composition of any of claims 254-263, wherein the synthetic or serum-free medium comprises albumin.

265. The synthetic medium or serum-free medium contains albumin, glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and trace element moiety Ag ＋ , Al ３＋ , Ba ２＋ , Cd ２＋ , Co ２＋ , Cr ３＋ , Ge ４＋ , Se ４＋ , Br, T, Mn ２＋ , P, Si ４＋ , V ５＋ , Mo ６＋ , Ni ２＋ , Rb ＋ , Sn ２＋ , and Zr ４＋ and one or more components selected from the group consisting of compounds containing:

266. 266. The TIL or expanded TIL composition of any of claims 254-265, wherein the synthetic or serum-free medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

267. The TIL or expanded TIL composition of any of claims 254-266, wherein the synthetic or serum-free medium has a total serum replacement concentration (volume %) of from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the cell culture medium.

268. The TIL or expanded TIL composition of any of claims 254-267, wherein the synthetic or serum-free medium has a total serum replacement concentration of about 3%, about 5%, or about 10% of the total volume of the cell culture medium.

269. The TIL or expanded TIL composition of any of claims 254-268, wherein the synthetic or serum-free medium further comprises glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM.

270. 270. The TIL or expanded TIL composition of any of claims 254-269, wherein said synthetic or serum-free medium further comprises glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

271. 271. The method of any of claims 254 to 270, wherein said synthetic or serum-free medium further comprises 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. The TIL or expanded TIL composition described above.

272. 272. The TIL or expanded TIL composition of any of claims 254-271, wherein said synthetic or serum-free medium further comprises 2-mercaptoethanol at a concentration of about 55 mM.

273. 273. The TIL or expanded TIL composition of any of claims 254-272, wherein said synthetic or serum-free medium comprises a synthetic medium described in International PCT Publication No. WO / 1998 / 030679.

274. The synthetic medium or serum-free medium may contain glycine in the range of about 5-200 mg / L, L-histidine in the range of about 5-250 mg / L, L-isoleucine in the range of about 5-300 mg / L, L-methionine in the range of about 5-200 mg / L, L-phenylalanine in the range of about 5-400 mg / L, L-proline in the range of about 1-1000 mg / L, L-hydroxyproline in the range of about 1-45 mg / L, L-serine in the range of about 1-250 mg / L, L-threonine in the range of about 10-500 mg / L, L-tryptophan in the range of about 2-110 mg / L, L-tyrosine in the range of about 3-175 mg / L, 274. The TIL or expanded TIL composition of any of claims 254-273, comprising L-valine in the range of about 5-500 mg / L, thiamine in the range of about 1-20 mg / L, reduced glutathione in the range of about 1-20 mg / L, L-ascorbic acid-2-phosphate in the range of about 1-200 mg / L, iron-saturated transferrin in the range of about 1-50 mg / L, insulin in the range of about 1-100 mg / L, sodium selenite in the range of about 0.000001-0.0001 mg / L, and / or albumin (e.g., AlbuMAX® I) in the range of about 5000-50,000 mg / L.

275. The TIL or expanded TIL composition of any of claims 254-274, wherein said synthetic or serum-free medium comprises one or more of the non-trace element moieties at the concentration ranges listed in the column under the heading "Concentration Range in 1x Medium" in Table A provided herein.

276. The TIL or expanded TIL composition of any of claims 254-275, wherein the osmolarity of the synthetic or serum-free medium is about 260-350 mOsmol.

277. 277. The TIL or expanded TIL composition of any of claims 254-276, wherein the synthetic or serum-free medium further comprises about 3.7 g / L, or about 2.2 g / L, of sodium bicarbonate.

278. The TIL or expanded TIL composition of any of claims 254-277, wherein the synthetic or serum-free medium further comprises L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA, final concentration of about 100 μM), and / or 2-mercaptoethanol (final concentration of about 100 μM).

279. 279. The TIL or expanded TIL composition of any of claims 254-278, wherein the synthetic or serum-free medium in the first and / or second gas permeable container lacks β-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

280. The cell culture medium is CTS OpTmizer T-Cell Expansion 280. The TIL or expanded TIL composition of any of claims 254-279, comprising SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

281. 281. The TIL or expanded TIL composition of any of claims 254-280, wherein the cell culture medium comprises CTS™ OpTmizer™ T-Cell Expansion Basal Medium supplemented with CTS™ OpTmizer™ T-Cell Expansion Supplement (26mL / L), and 3% CTS™ Immune Cell SR, and 2mM Glutamax, and optionally further comprising 6,000 IU / mL of IL-2.

282. 281. The TIL or expanded TIL composition of any of claims 254-280, wherein the cell culture medium comprises CTS™ OpTmizer™ T-Cell Expansion Supplement (26mL / L), and CTS™ OpTmizer™ T-Cell Expansion Basal Medium supplemented with 3% CTS™ Immune Cell SR, 2mM Glutamax, and optionally further comprising 3,000 IU / mL of IL-2.

283. The TIL or expanded TIL composition of any of claims 254-282, wherein the TIL population is a therapeutic TIL population.

284. The TIL or expanded TIL composition of any of claims 254-283, wherein the therapeutic TIL population exhibits elevated serum IFN-γ, wherein the elevated IFN-γ is greater than 200 pg / ml, greater than 250 pg / ml, greater than 300 pg / ml, greater than 350 pg / ml, greater than 400 pg / ml, greater than 450 pg / ml, greater than 500 pg / ml, greater than 550 pg / ml, greater than 600 pg / ml, greater than 650 pg / ml, greater than 700 pg / ml, greater than 750 pg / ml, greater than 800 pg / ml, greater than 850 pg / ml, greater than 900 pg / ml, greater than 950 pg / ml, or greater than 1000 pg / ml.

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