Methods for generating tumor-infiltrating lymphocytes and their use in immunotherapy

Abstract

The present invention provides an improved and / or shortened method for expanding TIL and producing a therapeutic population of TIL, including a novel method for expanding a TIL population in a closed system, the method leading to improved efficacy, improved phenotype and increased metabolic health of TIL in a shorter period of time, while allowing reduction of microbial contamination and cost reduction. Such TIL can be used for therapeutic treatment regimens.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 775,954 filed on November 5, 2018 and U.S. Provisional Patent Application No. 62 / 903,585 filed on September 20, 2019, both of which are hereby incorporated by reference in their entirety. Background Art

[0003] The use of adoptive transfer of tumor infiltrating lymphocytes (TIL) to treat large (bulky), refractory cancers represents a powerful treatment method for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires a large number of TILs, and commercialization requires a robust and reliable method. Due to technical, logistical and regulatory issues of cell expansion, it is a challenge to achieve this. IL-2-based TIL expansion and subsequent "rapid expansion process (REP)" have become the preferred method for TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can produce a 1,000-fold expansion of TILs within a 14-day period, although it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) usually from multiple donors as feeder cells, anti-CD3 antibodies (OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs undergoing the REP procedure have produced successful adoptive cell therapy in melanoma patients after host immunosuppression. Current infusion acceptance parameters rely on the readout of the composition of the TILs (e.g., CD28, CD8 or CD4 positivity) and the fold expansion and viability of the REP product.

[0004] Current TIL manufacturing methods are limited by length, cost, sterility considerations and other factors described herein. There is an urgent need to provide TIL manufacturing methods and therapies based on such methods, characterized by improved manufacturing cost effectiveness and scalability and more effective anti-cancer phenotypes of TIL preparations for treating human patients in multiple clinical centers. The present invention meets this need by providing a novel TIL expansion process that includes antigen presenting feeder cells from the beginning of expansion to induce TIL for expansion, rather than using traditional REP pre-expansion steps, thereby allowing a significant reduction in the overall time of the expansion process. Summary of the invention

[0005] The present invention provides improved and / or shortened methods for expanding TILs and generating therapeutic TIL populations.

[0006] The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising:

[0007] (a) obtaining and / or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

[0008] (b) performing a priming first 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 (APCs), wherein the priming first expansion is performed in a container comprising a first gas permeable surface area, wherein the priming first expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

[0009] (c) performing a rapid second 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 in the rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas permeable surface area;

[0010] (d) harvesting the therapeutic TIL population obtained from step (c); and

[0011] (e) Transferring the harvested TIL population from step (d) to an infusion bag.

[0012] The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising:

[0013] (a) obtaining and / or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

[0014] (b) performing a primed first 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 comprising antigen presenting cells (APCs), wherein the primed first expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

[0015] (c) performing a rapid second 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 rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and

[0016] (d) harvesting the therapeutic TIL population obtained from step (c).

[0017] In some embodiments, "obtaining" means that the TILs used in the method and / or process can be directly derived from a sample (including from a surgical resection, a needle biopsy, a core biopsy, a small biopsy, or other sample) as part of a method and / or process step. In some embodiments, "accepting" means that the TILs used in the method and / or process can be indirectly derived from a sample (including from a surgical resection, a needle biopsy, a core biopsy, a small biopsy, or other sample) and then used in the method and / or process (e.g., when step (a) starts with TILs that have been derived from a sample by a separate process not included in part (a), such TILs can be referred to as "accepting").

[0018] In some embodiments of the method, in step (b), the cell culture medium further comprises antigen presenting cells (APCs), and wherein 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).

[0019] The present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising:

[0020] (a) performing a priming first expansion to produce a second TIL population by culturing a first TIL population in a cell culture medium comprising IL-2, optionally OKT-3, and optionally antigen presenting cells (APCs), wherein the first TIL population can be obtained by processing a tumor sample from a tumor resected from a subject into a plurality of tumor fragments, wherein the priming first expansion is performed in a container comprising a first gas permeable surface area, wherein the priming first expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

[0021] (b) performing a rapid second expansion to produce a third TIL population by contacting the second TIL population with a cell culture medium of the second TIL population containing additional IL-2, OKT-3, and APCs, wherein the number of APCs in the rapid second expansion is at least twice the number of APCs in step (a), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas permeable surface area; and

[0022] (c) harvesting the therapeutic TIL population obtained from step (b).

[0023] The present invention also provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising:

[0024] (a) performing a primed first 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 comprising antigen presenting cells (APCs), wherein the primed first expansion is performed for a first period of about 1 to 7 / 8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

[0025] (b) performing a rapid second 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 rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and

[0026] (c) harvesting the therapeutic TIL population obtained from step (b).

[0027] In some embodiments of the method, in step (a), the cell culture medium further comprises antigen presenting cells (APCs), and wherein 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).

[0028] In some embodiments, the ratio of the number of APCs in the rapid second amplification to the number of APCs in the priming first amplification is selected from the range of about 1.5:1 to about 20:1.

[0029] In some embodiments, the ratio of the number of APCs in the rapid second amplification to the number of APCs in the priming first amplification is in the range of about 1.5:1 to about 10:1.

[0030] In some embodiments, the ratio of the number of APCs in the rapid second amplification to the number of APCs in the priming first amplification is in the range of about 2:1 to about 5:1.

[0031] In some embodiments, the ratio of the number of APCs in the rapid second amplification to the number of APCs in the priming first amplification is in the range of about 2:1 to about 3:1.

[0032] In some embodiments, the ratio of the number of APCs in the rapid second amplification to the number of APCs in the priming first amplification is about 2:1.

[0033] In some embodiments, the number of APCs in the priming first expansion is selected from about 1.0×10 6 APC / cm 2 About 4.5×10 6 APC / cm 2 and wherein the number of APCs in the rapid second expansion is selected from about 2.5×10 6 APC / cm 2 About 7.5×10 6 APC / cm 2 range.

[0034] In some embodiments, the number of APCs in the priming first expansion is selected from about 1.5×10 6 APC / cm 2 About 3.5×10 6 APC / cm 2 and wherein the number of APCs in the rapid second expansion is selected from about 3.5×10 6 APC / cm 2 About 6.0×10 6 APC / cm 2 range.

[0035] In some embodiments, the number of APCs in the priming first expansion is selected from about 2.0×10 6 APC / cm 2 About 3.0×10 6 APC / cm 2 and wherein the number of APCs in the rapid second expansion is selected from about 4.0×10 6 APC / cm 2 About 5.5×10 6 APC / cm 2 range.

[0036] In some embodiments, the number of APCs in the priming first expansion is selected from about 1×10 8 APC is about 3.5×10 8 APCs, and wherein the number of APCs in the rapid second expansion is selected from about 3.5×10 8 APC is about 1×10 9 The range of APCs.

[0037] In some embodiments, the number of APCs in the priming first expansion is selected from about 1.5×10 8 APC is about 3×10 8 APCs, and wherein the number of APCs in the rapid second expansion is selected from about 4×10 8 APC is about 7.5×10 8 The range of APCs.

[0038] In some embodiments, the number of APCs in the priming first expansion is selected from about 2×10 8 APC is about 2.5×10 8 APCs, and wherein the number of APCs in the rapid second expansion is selected from about 4.5×10 8 APC is about 5.5×10 8 The range of APCs.

[0039] In some embodiments, about 2.5×10 8 APCs were added to the primary amplification and 5×10 8 APCs were added to the rapid second amplification.

[0040] 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 1.5:1 to about 100:1.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In some embodiments, the second TIL population is at least 50 times greater in number than the first TIL population.

[0046] In some embodiments, the method comprises, after the step of harvesting the therapeutic TIL population, performing the following additional steps:

[0047] The harvested therapeutic TIL population was transferred to an infusion bag.

[0048] In some embodiments, the plurality of tumor fragments are distributed in a plurality of separate containers, in each of the separate containers, the second TIL population is obtained from the first TIL population in the priming first expansion step, and the third TIL population is obtained from the second TIL population in the rapid second expansion step, and wherein the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to produce a harvested TIL population.

[0049] In some embodiments, the plurality of separate containers comprises at least two separate containers.

[0050] In some embodiments, the plurality of separate containers comprises two to twenty separate containers.

[0051] In some embodiments, the plurality of separate containers comprises two to ten separate containers.

[0052] In some embodiments, the plurality of separate containers comprises two to five separate containers.

[0053] In some embodiments, each of the separate containers comprises a first gas permeable surface area.

[0054] In some embodiments, the plurality of tumor fragments are distributed in a single container.

[0055] In some embodiments, the single container includes a first gas permeable surface region.

[0056] In some embodiments, in the priming first expansion step, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered on the first gas permeable surface area at an average thickness of about one cell layer to about three cell layers.

[0057] In some embodiments, in the priming first expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

[0058] In some embodiments, in the priming first expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 2 cell layers.

[0059] In some embodiments, in the rapid second expansion step, the APCs are layered on the first gas permeable surface area at a thickness of about 3 cell layers to about 5 cell layers.

[0060] In some embodiments, the rapid second expanded APCs are layered on the first gas permeable surface area at a thickness of about 3.5 cell layers to about 4.5 cell layers.

[0061] In some embodiments, the rapid second expanded APCs are layered on the first gas permeable surface area at a thickness of about 4 cell layers.

[0062] In some embodiments, in the priming first amplification step, the priming first amplification is performed in a first container comprising a first gas permeable surface area, and in the rapid second amplification step, the rapid second amplification is performed in a second container comprising a second gas permeable surface area.

[0063] In some embodiments, the second container is larger than the first container.

[0064] In some embodiments, in the priming first expansion step, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered on the first gas permeable surface area at an average thickness of about one cell layer to about three cell layers.

[0065] In some embodiments, in the priming first expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

[0066] In some embodiments, in the priming first expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 2 cell layers.

[0067] In some embodiments, the rapidly second expanded APCs are layered on the second gas permeable surface area at an average thickness of about 3 cell layers to about 5 cell layers.

[0068] In some embodiments, the rapidly second expanded APCs are layered on the second gas permeable surface area at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

[0069] In some embodiments, in the rapid second expansion step, the APCs are layered on the second gas permeable surface area at an average thickness of about 4 cell layers.

[0070] In some embodiments, the priming first amplification is performed on a first TIL population in each container, and the rapid second amplification is performed on the second TIL population generated from such first TIL population in the same container.

[0071] In some embodiments, each container includes a first gas permeable surface region.

[0072] In some embodiments, in the priming first expansion step, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered on the first gas permeable surface area at an average thickness of about one cell layer to about three cell layers.

[0073] In some embodiments, in the priming first expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

[0074] In some embodiments, in the priming first expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 2 cell layers.

[0075] In some embodiments, in the rapid second expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 3 cell layers to about 5 cell layers.

[0076] In some embodiments, in the rapid second expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

[0077] In some embodiments, in the rapid second expansion step, the APCs are layered on the first gas permeable surface area at an average thickness of about 4 cell layers.

[0078] In some embodiments, for each container in which the priming first expansion is performed on the first TIL population, in the priming first expansion step, the first container includes a first surface area, the cell culture medium contains antigen presenting cells (APCs), and the APCs are layered on the first gas permeable surface area, and wherein the ratio of the average number of APC layers layered in the priming first expansion step to the average number of APC layers layered in the rapid second expansion step is selected from the range of about 1:1.1 to about 1:10.

[0079] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.2 to about 1:8.

[0080] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.3 to about 1:7.

[0081] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.4 to about 1:6.

[0082] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.5 to about 1:5.

[0083] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.6 to about 1:4.

[0084] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.7 to about 1:3.5.

[0085] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.8 to about 1:3.

[0086] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is selected from the range of about 1:1.9 to about 1:2.5.

[0087] In some embodiments, the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is about 1:2.

[0088] In some embodiments, after 2 to 3 days in the rapid second expansion step, the cell culture medium is supplemented with additional IL-2.

[0089] In some embodiments, the method further comprises cryopreserving the harvested TIL population in the step of harvesting the therapeutic TIL population using a cryopreservation process.

[0090] In some embodiments, the method further comprises the step of cryopreserving the infusion bag.

[0091] In some embodiments, the cryopreservation process is performed using a 1:1 ratio of harvested TIL population to cryopreservation medium.

[0092] In some embodiments, the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).

[0093] In some embodiments, the PBMCs are irradiated and allogeneic.

[0094] In some embodiments, in the priming first expansion step, the cell culture medium comprises peripheral blood mononuclear cells (PBMCs), and wherein the total number of PBMCs added to the cell culture medium in the priming first expansion step is about 2.5×10 8 indivual.

[0095] In some embodiments, in the rapid second expansion step, the antigen presenting cells (APCs) in the cell culture medium are peripheral blood mononuclear cells (PBMCs), and wherein the total number of PBMCs added to the cell culture medium in the rapid second expansion step is about 5×10 8 indivual.

[0096] In some embodiments, the antigen presenting cell is an artificial antigen presenting cell.

[0097] In some embodiments, the harvesting in the step of harvesting the therapeutic TIL population is performed using a membrane-based cell processing system.

[0098] In some embodiments, the harvesting in the step of harvesting the therapeutic TIL population is performed using the LOVO cell processing system.

[0099] In some embodiments, in the priming first amplification step, the plurality of fragments comprises about 60 fragments per container, wherein each fragment has about 27 mm 3 volume.

[0100] In some embodiments, the plurality of segments comprises about 30 to about 60 segments having a total volume of about 1300 mm 3 To about 1500mm 3 .

[0101] In some embodiments, the plurality of segments comprises about 50 segments having a total volume of about 1350 mm 3 .

[0102] In some embodiments, the plurality of fragments includes about 50 fragments having a total mass of about 1 gram to about 1.5 grams.

[0103] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

[0104] In some embodiments, the IL-2 concentration is about 10,000 IU / mL to about 5,000 IU / mL.

[0105] In some embodiments, the IL-2 concentration is about 6,000 IU / mL.

[0106] In some embodiments, in the step of transferring the harvested therapeutic TIL population to an infusion bag, the infusion bag is an infusion bag containing HypoThermosol.

[0107] In some embodiments, the cryopreservation medium comprises dimethyl sulfoxide (DMSO).

[0108] In some embodiments, the cryopreservation medium comprises 7% to 10% DMSO.

[0109] In some embodiments, the first period of time of the priming first amplification step and the second period of time of the rapid second amplification step are each individually performed over a period of 5 days, 6 days, or 7 days.

[0110] In some embodiments, the first period of time of the priming first amplification step is performed over a period of 5 days, 6 days, or 7 days.

[0111] In some embodiments, the second period of time of the rapid second amplification step is performed over a period of 7 days, 8 days, or 9 days.

[0112] In some embodiments, the first period of time of the priming first amplification step and the second period of time of the rapid second amplification step are each individually performed over a period of 7 days.

[0113] In some embodiments, the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed over a period of about 14 days to about 16 days.

[0114] In some embodiments, the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed over a period of about 15 days to about 16 days.

[0115] In some embodiments, the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed within a period of about 14 days.

[0116] In some embodiments, the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed within a period of about 15 days.

[0117] In some embodiments, the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed over a period of about 16 days.

[0118] In some embodiments, the method further comprises the step of cryopreserving the harvested therapeutic TIL population using a cryopreservation process, wherein the steps of priming first expansion to harvesting the therapeutic TIL population and cryopreservation are performed within 16 days or less.

[0119] In some embodiments, the therapeutic TIL population harvested in the step of harvesting the therapeutic TIL population comprises sufficient TILs for a therapeutically effective dose of the TILs.

[0120] In some embodiments, a sufficient number of TILs for a therapeutically effective dose is about 2.3×10 10 to about 13.7×10 10 indivual.

[0121] In some embodiments, the third TIL population in the rapid second expansion step provides increased efficacy, increased interferon-γ production, and / or increased polyclonality.

[0122] In some embodiments, the third TIL population in the rapid second expansion step provides at least 1-fold to 5-fold or more interferon-γ production compared to TILs prepared by a process longer than 18 days.

[0123] In some embodiments, the effector T cells and / or central memory T cells obtained from the third TIL population in the rapid second expansion step exhibit increased expression of CD8 and CD28 relative to the effector T cells and / or central memory T cells obtained from the second TIL population in the priming first expansion step.

[0124] In some embodiments, the therapeutic TIL population from the step of harvesting the therapeutic TIL population is infused into a patient.

[0125] The present invention also provides a method for treating a subject having cancer, the method comprising administering expanded tumor infiltrating lymphocytes (TILs), the method comprising:

[0126] (a) obtaining and / or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

[0127] (b) performing a priming first 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 (APCs), wherein the priming first expansion is performed in a container comprising a first gas permeable surface area, wherein the priming first expansion is performed for about 1 to 7 / 8 days to obtain the second TIL population, wherein the second TIL population is at least 50 times greater in number than the first TIL population;

[0128] (c) performing a rapid second 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 rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas permeable surface area;

[0129] (d) harvesting the therapeutic TIL population obtained from step (c);

[0130] (e) transferring the harvested TIL population from step (d) to an infusion bag; and

[0131] (f) administering a therapeutically effective dose of the TILs from step (e) to the subject.

[0132] In some embodiments, a sufficient number of TILs for administering a therapeutically effective dose in step (f) is about 2.3×10 10 to about 13.7×10 10 indivual.

[0133] In some embodiments, the antigen presenting cell (APC) is a PBMC.

[0134] In some embodiments, the patient has been administered a myeloablative lymphocyte depletion regimen prior to administering the therapeutically effective dose of TIL cells in step (f).

[0135] In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administering a dose of 60 mg / m 2 Cyclophosphamide was administered daily for two days followed by 25 mg / m 2 / day of fludarabine for five days in steps.

[0136] In some embodiments, the method further comprises the step of treating the patient with a high-dose IL-2 regimen beginning on the day after administering the TIL cells to the patient in step (f).

[0137] 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 eight hours until tolerated.

[0138] In some embodiments, the third TIL population in step (b) provides increased efficacy, increased interferon-γ production, and / or increased polyclonality.

[0139] In some embodiments, the third TIL population in step (c) provides at least 1-fold to 5-fold or more interferon-γ production compared to TILs prepared by a process longer than 16 days.

[0140] In some embodiments, the effector T cells and / or central memory T cells obtained from the third TIL population in step (c) exhibit increased expression of CD8 and CD28 relative to the effector T cells and / or central memory T cells obtained from the second cell population in step (b).

[0141] In some embodiments, the cancer is a solid tumor.

[0142] 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 papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma.

[0143] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0144] In some embodiments, the cancer is melanoma.

[0145] In some embodiments, the cancer is HNSCC.

[0146] In some embodiments, the cancer is cervical cancer.

[0147] In some embodiments, the cancer is NSCLC.

[0148] In some embodiments, the cancer is glioblastoma (including GBM).

[0149] In some embodiments, the cancer is gastrointestinal cancer.

[0150] In some embodiments, the cancer is a hypermutated cancer.

[0151] In some embodiments, the cancer is a pediatric hypermutated cancer.

[0152] In some embodiments, the container is a closed container.

[0153] In some embodiments, the container is a G container.

[0154] In some embodiments, the container is GREX-10.

[0155] In some embodiments, the sealed container comprises GREX-100.

[0156] In some embodiments, the sealed container comprises GREX-500.

[0157] The present invention also provides therapeutic tumor infiltrating lymphocyte (TIL) populations produced by the methods as disclosed herein.

[0158] The present invention also provides a therapeutic tumor infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, wherein the therapeutic TIL population provides increased efficacy, increased interferon-γ production and / or increased polyclonality.

[0159] In some embodiments, a therapeutic TIL population as disclosed herein provides increased interferon-γ production.

[0160] In some embodiments, a therapeutic TIL population as disclosed herein provides increased polyclonality.

[0161] In some embodiments, a therapeutic TIL population as disclosed herein provides increased efficacy.

[0162] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 1-fold more interferon-γ compared to TIL prepared by a process longer than 16 days.

[0163] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 2-fold more interferon-γ compared to TIL prepared by a process longer than 16 days.

[0164] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 3-fold more interferon-γ compared to TIL prepared by a process longer than 16 days.

[0165] In some embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population, wherein the therapeutic TIL population is capable of producing at least 1-fold more interferon-γ compared to TIL prepared by a process wherein the first expansion of the TIL is performed without any added antigen presenting cells (APCs).

[0166] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 2-fold more interferon-γ compared to TIL prepared by a process wherein the first expansion of the TIL is performed without any added APCs.

[0167] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 3-fold more interferon-γ compared to TIL prepared by a process wherein the first expansion of the TIL is performed without any added APCs.

[0168] In some embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population, wherein the therapeutic TIL population is capable of producing at least 1-fold more interferon-γ compared to TIL prepared by a process wherein the first expansion of the TIL is performed without any added OKT3.

[0169] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 2-fold more interferon-γ compared to TIL prepared by a process wherein the first expansion of the TIL is performed without any added OKT3.

[0170] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 3-fold more interferon-γ compared to TIL prepared by a process wherein the first expansion of the TIL is performed without any added OKT3.

[0171] In some embodiments, the present invention provides a therapeutic tumor infiltrating lymphocyte (TIL) population, wherein the therapeutic TIL population is capable of producing at least 1-fold more interferon-γ compared to TIL prepared by a process in which the first expansion of the TIL is performed in the absence of added antigen presenting cells (APCs) and in the absence of added OKT3.

[0172] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 2-fold more interferon-γ compared to TIL prepared by a process in which the first expansion of the TIL is performed without added antigen presenting cells (APCs) and without added OKT3.

[0173] In some embodiments, a therapeutic TIL population as described herein is capable of producing at least 3-fold more interferon-γ compared to TIL prepared by a process in which the first expansion of the TIL is performed without added antigen presenting cells (APCs) and without added OKT3.

[0174] The present invention also provides a tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described herein and a pharmaceutically acceptable carrier.

[0175] The present invention also provides a sterile infusion bag, comprising the TIL composition as described herein.

[0176] The present invention also provides a cryopreserved preparation of a therapeutic TIL population as described herein.

[0177] The present invention also provides a tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described herein and a cryopreservation medium.

[0178] In some embodiments, the cryopreservation medium contains DMSO.

[0179] In some embodiments, the cryopreservation medium contains 7% to 10% DMSO.

[0180] The present invention also provides a cryopreserved preparation of a TIL composition as described herein.

[0181] In some embodiments, the tumor infiltrating lymphocyte (TIL) composition as described herein is used as a medicament.

[0182] In some embodiments, the tumor infiltrating lymphocyte (TIL) compositions described herein are used to treat cancer.

[0183] In some embodiments, the tumor infiltrating lymphocyte (TIL) compositions described herein are used to treat solid tumor cancers.

[0184] In some embodiments, the tumor infiltrating lymphocyte (TIL) composition as described herein is used to treat 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 papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma.

[0185] In some embodiments, the tumor infiltrating lymphocyte (TIL) composition as described herein is used to treat a cancer selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0186] In some embodiments, a TIL composition as described herein is used to treat cancer, wherein the cancer is melanoma.

[0187] In some embodiments, a TIL composition as described herein is used to treat cancer, wherein the cancer is HNSCC.

[0188] In some embodiments, the TIL composition as described herein is used to treat cancer, wherein the cancer is cervical cancer.

[0189] In some embodiments, the TIL composition as described herein is used to treat cancer, wherein the cancer is NSCLC.

[0190] In some embodiments, a TIL composition as described herein is used to treat cancer, wherein the cancer is glioblastoma (including GBM).

[0191] In some embodiments, the TIL composition as described herein is used to treat cancer, wherein the cancer is gastrointestinal cancer.

[0192] In some embodiments, a TIL composition as described herein is used to treat cancer, wherein the cancer is a hypermutated cancer.

[0193] In some embodiments, a TIL composition as described herein is used to treat cancer, wherein the cancer is a pediatric hypermutated cancer.

[0194] In some embodiments, the present invention provides a method for treating a subject's cancer using a tumor-infiltrating lymphocyte (TIL) composition as described herein, 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 papilloma virus, 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, 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 a gastrointestinal cancer. In some embodiments, the cancer is a hypermutation cancer. In some embodiments, the cancer is a pediatric hypermutation cancer.

[0195] In some embodiments, a tumor infiltrating lymphocyte (TIL) composition as described herein is used in a method for treating a subject's cancer, 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 papilloma virus, 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.

[0196] The present invention also provides a method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective dose of the tumor infiltrating lymphocyte (TIL) composition as described herein.

[0197] In some embodiments, the cancer is a solid tumor.

[0198] 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 papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma.

[0199] 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 mutation cancer. In some embodiments, the cancer is a pediatric high mutation cancer.

[0200] The present invention also provides a method for expanding T cells, the method comprising:

[0201] (a) performing a primed first expansion of a first T cell population obtained from a donor by culturing the first T cell population to achieve growth of the first T cell population and to prime the activation of the first T cell population;

[0202] (b) after the activation of the first T cell population triggered in step (a) begins to decline, rapidly expanding the first T cell population by culturing the first T cell population for a second time, so as to achieve growth of the first T cell population and strengthen the activation of the first T cell population to obtain a second T cell population; and

[0203] (c) harvesting the second T cell population.

[0204] In some embodiments, the priming first amplification of step (a) is performed over a period of up to 7 days.

[0205] In some embodiments, the rapid second amplification of step (b) is performed over a period of up to 11 days.

[0206] In some embodiments, the rapid second amplification of step (b) is performed over a period of up to 9 days.

[0207] In some embodiments, the priming first amplification of step (a) is performed over a period of 7 days and the rapid second amplification of step (b) is performed over a period of 9 days.

[0208] In some embodiments, the priming first amplification of step (a) is performed over a period of up to 8 days.

[0209] In some embodiments, the rapid second amplification of step (b) is performed over a period of up to 8 days.

[0210] In some embodiments, the priming first amplification of step (a) is performed over a period of 8 days and the rapid second amplification of step (b) is performed over a period of 8 days.

[0211] In some embodiments of the method, in step (a), the first population of T cells is cultured in a first culture medium comprising OKT-3 and IL-2.

[0212] In some embodiments, the first culture medium comprises OKT-3, IL-2, and antigen presenting cells (APCs).

[0213] In some embodiments of the method, 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).

[0214] In some embodiments of the method, in step (a), the first population of T cells is cultured in a first culture medium in a container comprising a first gas permeable surface, wherein the first culture medium comprises optionally OKT-3, IL-2, and optionally a first population of antigen presenting cells (APCs), wherein the first population of APCs is exogenous to the donor of the first population of T cells and the first population of APCs is layered on the first gas permeable surface, wherein in step (b), the first population of T cells is cultured in a second culture medium in the container, wherein the second culture medium comprises OKT-3, IL-2, and a second population of APCs, wherein the second population of APCs is exogenous to the donor of the first population of T cells and the second population of APCs is layered on the first gas permeable surface, and wherein the second population of APCs is larger than the first population of APCs.

[0215] In some embodiments, the ratio of the number of APCs in the second population of APCs to the number of APCs in the first population of APCs is about 2:1.

[0216] In some embodiments, the number of APCs in the first APC population is about 2.5 x 10 8 and the number of APCs in the second APC population is about 5x10 8 indivual.

[0217] In some embodiments of the method, in step (a), the first population of APCs is layered on the first gas permeable surface with an average thickness of 2 layers of APCs.

[0218] In some embodiments of the method, in step (b), the second APC population is layered on the first gas permeable surface at an average thickness selected from the range of 4 to 8 APC layers.

[0219] 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.

[0220] In some embodiments, the APCs are peripheral blood mononuclear cells (PBMCs).

[0221] In some embodiments, the APCs comprise PBMCs, wherein the PBMCs are irradiated and exogenous to the donor of the first T cell population.

[0222] In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs).

[0223] In some embodiments, the T cells are bone marrow infiltrating lymphocytes (MIL).

[0224] In some embodiments, the T cells are peripheral blood lymphocytes (PBL).

[0225] In some embodiments, the cell culture medium is a defined medium and / or a serum-free medium.

[0226] In some embodiments, the defined medium comprises (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0227] In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and / or serum replacement.

[0228] In some embodiments, the basal cell culture medium includes but is not limited to 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 TMT cell expansion Xeno-free medium, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

[0229] In some embodiments, the serum supplement or serum replacement is selected from the group consisting of: CTS TM OpTmizer T Cell Expansion Serum Supplement and CTS TM Immune cell serum replacement.

[0230] In some embodiments, the cell culture medium comprises one or more albumins or albumin substitutes.

[0231] In some embodiments, the cell culture medium comprises one or more amino acids.

[0232] In some embodiments, the cell culture medium comprises one or more vitamins, one or more transferrins or transferrin substitutes.

[0233] In some embodiments, the cell culture medium comprises one or more antioxidants, one or more insulins or insulin substitutes.

[0234] In some embodiments, the cell culture medium comprises one or more collagen precursors, one or more antibiotics, and one or more trace elements.

[0235] In some embodiments, the cell culture medium comprises albumin.

[0236] 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 trace element Ag-containing portion. + 、Al 3+ , Ba 2+ 、Cd 2+ 、Co 2+ Cr 3+ ,Ge 4+ 、Se 4+ ,Br,T,Mn 2+ 、P、Si 4+ 、V5+ 、Mo 6+ 、Ni 2+ , Rb + Sn 2+ and Zr 4+ of compounds.

[0237] In some embodiments, the cell culture medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0238] In some embodiments, the cell culture medium comprises a total serum replacement concentration of 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 (vol %).

[0239] In some embodiments, the cell culture medium comprises a total serum replacement concentration of about 3%, about 5%, or about 10% of the total volume of the cell culture medium.

[0240] In some embodiments, the cell culture medium further comprises glutamine (i.e., glutamine) 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. ).

[0241] In some embodiments, the cell culture medium further comprises glutamine at a concentration of about 2 mM (i.e. ).

[0242] 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.

[0243] In some embodiments, the cell culture medium further comprises 2-mercaptoethanol at a concentration of about 55 mM.

[0244] In some embodiments, the cell culture medium comprises a defined medium described in International PCT Publication No. WO / 1998 / 030679.

[0245] 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 10 to 500 mg / L, L-threonine in the range of about 2 to 110 mg / L, L-threonine in the range of about 10 to 500 mg / L, L-hydroxyproline in the range of about 10 to 250 mg / L, L-threonine in the range of about 10 to 500 mg / L, L-hydroxyproline in the range of about 10 to 250 mg / L, L-threonine in the range of about 10 to 25 ... L-tryptophan in the range of about 3 to 175 mg / L, L-tyrosine in the range of about 5 to 500 mg / L, L-valine in the range of about 1 to 20 mg / L, thiamine in the range of about 1 to 20 mg / L, reduced glutathione in the range of about 1 to 200 mg / L, L-ascorbic acid-2-phosphate in the range of about 1 to 50 mg / L, iron-saturated transferrin in the range of about 1 to 100 mg / L, insulin in the range of about 0.000001 to 0.0001 mg / L, sodium selenite in the range of about 5000 to 50,000 mg / L (e.g., I).

[0246] In some embodiments, the cell culture medium comprises one or more non-trace element portion components of a defined medium, said components being present in a concentration range listed in the column entitled "Concentration Range in IX Medium" in Table A provided herein.

[0247] In some embodiments, the osmolarity of the cell culture medium is between about 260 and 350 mOsmol.

[0248] In some embodiments, the cell culture medium further comprises about 3.7 g / L or about 2.2 g / L sodium bicarbonate.

[0249] In some embodiments, the cell culture 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).

[0250] In some embodiments, the cell culture medium in the first gas permeable container and / or the second gas permeable container lacks beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0251] In some embodiments, the cell culture medium comprises CTS OpTmizer T Cell Expansion SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

[0252] In some embodiments, the cell culture medium comprises a cell culture medium supplemented with CTS TM OpTmizer TM CTS of T Cell Expansion Supplement (26mL / L) TM OpTmizer TM T cell expansion basal medium and 3% CTS TM Immune Cell SR and 2 mM Glutamax, optionally also containing 6,000 IU / mL of IL-2.

[0253] In some embodiments, the cell culture medium comprises a cell culture medium supplemented with CTS TM OpTmizer TM CTS of T Cell Expansion Supplement (26mL / L) TM OpTmizer TM T cell expansion basal medium and 3% CTS TM Immune Cell SR, 2 mM Glutamax, and optionally also contains 3,000 IU / mL of IL-2.

[0254] The present invention also provides a tumor infiltrating lymphocyte (TIL) composition, comprising:

[0255] i) a therapeutic tumor infiltrating lymphocyte (TIL) population, and

[0256] ii) a defined or serum-free medium optionally comprising (optionally recombinant) transferrin, (optionally recombinant) insulin and (optionally recombinant) albumin.

[0257] The present invention also provides an expanded tumor infiltrating lymphocyte (TIL) composition, comprising:

[0258] i) a therapeutic tumor infiltrating lymphocyte (TIL) population, and

[0259] ii) a defined or serum-free medium optionally comprising (optionally recombinant) transferrin, (optionally recombinant) insulin and (optionally recombinant) albumin.

[0260] In some embodiments, the defined medium or serum-free medium comprises (optionally recombinant) transferrin, (optionally recombinant) insulin, and (optionally recombinant) albumin.

[0261] In some embodiments, the defined medium or serum-free medium comprises a basal cell culture medium and a serum supplement and / or serum replacement.

[0262] In some embodiments, the basal cell culture medium includes but is not limited to 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), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

[0263] In some embodiments, the serum supplement or serum replacement is selected from the group consisting of: CTS TM OpTmizer T Cell Expansion Serum Supplement and CTS TM Immune cell serum replacement.

[0264] In some embodiments, the defined medium or serum-free medium comprises one or more albumins or albumin substitutes.

[0265] In some embodiments, the defined medium or serum-free medium comprises one or more amino acids.

[0266] In some embodiments, the defined medium or serum-free medium comprises one or more vitamins, one or more transferrins or transferrin substitutes.

[0267] In some embodiments, the defined medium or serum-free medium comprises one or more antioxidants, one or more insulins or insulin substitutes.

[0268] In some embodiments, the defined medium or serum-free medium comprises one or more collagen precursors, one or more antibiotics, and one or more trace elements.

[0269] In some embodiments, the defined medium or serum-free medium comprises albumin.

[0270] In some embodiments, the defined medium or serum-free 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 portion of trace elements 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+ of compounds.

[0271] In some embodiments, the defined medium or serum-free medium further comprises L-glutamine, sodium bicarbonate and / or 2-mercaptoethanol.

[0272] In some embodiments, the defined medium or serum-free medium comprises a total serum replacement concentration of 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 (vol %).

[0273] In some embodiments, the defined medium or serum-free medium comprises a total serum replacement concentration of about 3%, about 5%, or about 10% of the total volume of the cell culture medium.

[0274] In some embodiments, the defined medium or serum-free medium further comprises glutamine (i.e., glutamine) 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. ).

[0275] In some embodiments, the defined medium or serum-free medium further comprises glutamine at a concentration of about 2 mM (i.e. ).

[0276] In some embodiments, the defined 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.

[0277] In some embodiments, the defined medium or serum-free medium further comprises 2-mercaptoethanol at a concentration of about 55 mM.

[0278] In some embodiments, the defined medium or serum-free medium comprises a defined medium described in International PCT Publication No. WO / 1998 / 030679.

[0279] In some embodiments, the defined medium or serum-free 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, and L-threonine in the range of about 2 to 1 10 mg / L of L-tryptophan, about 3 to 175 mg / L of L-tyrosine, about 5 to 500 mg / L of L-valine, about 1 to 20 mg / L of thiamine, about 1 to 20 mg / L of reduced glutathione, about 1 to 200 mg / L of L-ascorbic acid-2-phosphate, about 1 to 50 mg / L of iron-saturated transferrin, about 1 to 100 mg / L of insulin, about 0.000001 to 0.0001 mg / L of sodium selenite, and / or about 5000 to 50,000 mg / L of albumin (e.g., I).

[0280] In some embodiments, the defined medium or serum-free medium comprises one or more non-trace element portion components of a defined medium, which are present in a concentration range listed in the column entitled "Concentration Range in IX Medium" in Table A provided herein.

[0281] In some embodiments, the osmolarity of the defined medium or serum-free medium is between about 260 and 350 mOsmol.

[0282] In some embodiments, the defined medium or serum-free medium further comprises about 3.7 g / L or about 2.2 g / L sodium bicarbonate.

[0283] In some embodiments, the defined medium 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).

[0284] In some embodiments, the defined medium or serum-free medium in the first gas permeable container and / or the second gas permeable container lacks beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0285] In some embodiments, the cell culture medium comprises CTS OpTmizer T Cell Expansion SFM, 3% CTS Immune Cell Serum Replacement, 55 mM BME, and optionally glutamine.

[0286] In some embodiments, the cell culture medium comprises a cell culture medium supplemented with CTS TM OpTmizer TM CTS of T Cell Expansion Supplement (26mL / L) TM OpTmizer TM T cell expansion basal medium and 3% CTS TM Immune Cell SR and 2 mM Glutamax, optionally also containing 6,000 IU / mL of IL-2.

[0287] In some embodiments, the cell culture medium comprises a cell culture medium supplemented with CTS TM OpTmizer TM CTS of T Cell Expansion Supplement (26mL / L) TM OpTmizer TM T cell expansion basal medium and 3% CTS TM Immune Cell SR, 2 mM Glutamax, and optionally also contains 3,000 IU / mL of IL-2.

[0288] In some embodiments, the TIL population is a therapeutic TIL population.

[0289] In some embodiments, the therapeutic TIL population exhibits an increase in serum IFN-γ, wherein the increase in IFN-γ is greater than 200pg / ml, greater than 250pg / ml, greater than 300pg / ml, greater than 350pg / ml, greater than 400pg / ml, greater than 450pg / ml, greater than 500pg / ml, greater than 550pg / ml, greater than 600pg / ml, greater than 650pg / ml, greater than 700pg / ml, greater than 750pg / ml, greater than 800pg / ml, greater than 850pg / ml, greater than 900pg / ml, greater than 950pg / ml, or greater than 1000pg / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0290] Figures 1A-1C : A) shows a comparison of the 2A process (approximately 22 day process) and the 3rd generation process embodiment (approximately 14 to 16 day process) for TIL manufacturing. B) Diagram of an exemplary 3rd generation process provides an overview of steps A to F (approximately 14 to 16 day process). C) Diagram provides an overview of three exemplary 3rd generation processes and three process variations of steps A to F (approximately 14 to 16 day process).

[0291] Figure 2 : Provides an experimental flow chart for comparability between the 2nd generation (2A process) and the 3rd generation.

[0292] Figure 3 A-3C: A) L4054: Phenotypic characterization of TIL products at passages 2 and 3. B) L4055: Phenotypic characterization of TIL products at passages 2 and 3. C) M1085T: Phenotypic characterization of TIL products at passages 2 and 3.

[0293] Figure 4 A-4C: A) L4054: Memory marker analysis of TIL products at passages 2 and 3. B) L4055: Memory marker analysis of TIL products at passages 2 and 3. C) M1085T: Memory marker analysis of TIL products at passages 2 and 3.

[0294] Figure 5 : L4054 activation and exhaustion markers (A) gated on CD4+, (B) gated on CD8+.

[0295] Figure 6 : L4055 activation and exhaustion markers (A) gated on CD4+, (B) gated on CD8+.

[0296] Figure 7: IFNγ production (pg / mL): Passage 2 and 3 process for (A) L4054, (B) L4055 and (C) M1085T: Each bar represented here refers to the mean + SEM of IFNγ levels for stimulated, unstimulated and medium control. Optical density was measured at 450 nm.

[0297] Figure 8 : ELISA analysis of IL-2 concentration in cell culture supernatants: (A) L4054 and (B) L4055. Each bar represented here refers to the mean + SEM of IL-2 levels in spent culture medium. Optical density was measured at 450 nm.

[0298] Fig. 9 : Quantification of glucose and lactate (g / L) in spent media: (A) Glucose and (B) Lactate: In both tumor cell lines and both processes, a decrease in glucose was observed throughout REP expansion. In contrast, an increase in lactate was observed as expected. The decrease in glucose and increase in lactate between the 2nd and 3rd generation processes were comparable.

[0299] Figure 10: A) Quantification of L-glutamine in spent medium of L4054 and L4055. B) Quantification of Glutamax in spent medium of L4054 and L4055. C) Quantification of ammonia in spent medium of L4054 and L4055.

[0300] Fig.11 : Telomere length analysis. Relative telomere length (RTL) values ​​indicate the average telomere fluorescence per chromosome / genome during passages 2 and 3 using the DAKO kit relative to the telomere fluorescence per chromosome / genome in a control cell line (1301 leukemia cell line).

[0301] Fig.12 : Unique CDR3 sequence analysis of the final products of the 2nd and 3rd generation process TILs of L4054 and L4055. The columns show 1×10 collected from the harvest days of the 2nd generation (e.g., day 22) and the 3rd generation process (e.g., days 14 to 16). 6 The number of unique TCR B clone types identified per cell. Based on the number of unique peptide CDRs in the samples, generation 3 showed higher clonal diversity compared to generation 2.

[0302] Fig.13 : Frequency of unique CDR3 sequences in L4054 IL harvest final cell products (passage 2 (e.g., day 22) and passage 3 processes (e.g., days 14 to 16)).

[0303] Fig.14: Frequency of unique CDR3 sequences in the final cell products of L4055 TIL harvest (passage 2 (e.g., day 22) and passage 3 process (e.g., days 14 to 16)).

[0304] Fig.15 : Diversity index of the final products of the 2nd and 3rd generation process TILs of L4054 and L4055. The Shanon entropy diversity index is a more reliable and common comparative measure. The 3rd generation L4054 and L4055 showed slightly higher diversity compared to the 2nd generation.

[0305] Fig.16 : Day 7 - Cell counts starting at passage 3 REP Raw data are presented in Table 22 (see Example 5 below).

[0306] Fig.17 : Day 11 - Cell count raw data for REP initiation at Passage 2 and scale up at Passage 3 are presented in Table 22 (see Example 5 below).

[0307] Fig.18 : Day 16 - Raw data of cell counts for passage 2 scale-up and harvest 3 (eg, day 16) are presented in Table 23 (see Example 5 below).

[0308] Fig.19 : Day 22 - Raw cell count data for passage 2 harvest (e.g., day 22) are presented in Table 23 (see Example 5 below). For L4054 passage 2, post-LOVO counts were extrapolated to 4 flasks, as that was the total number studied. One flask was contaminated, and the extrapolated total = 6.67E+10.

[0309] Fig. 20 : Depicted on Figure 3 A, Raw data of flow cytometry results of 4A and 4B.

[0310] Fig.21 : Depicted on Figure 3 Raw data of flow cytometry results in C and 4C.

[0311] Fig. 22 : Depicted on Figure 5 and 6 Raw data of flow cytometry results.

[0312] Figure 23: Depicted on Figure 7 Raw data of the IFNγ production assay results for the L4054 sample.

[0313] Figure 24: Depicted on Figure 7 Raw data of the IFNγ production assay results for the L4055 sample.

[0314] Figure 25: Depicted on Figure 7 Raw data of the IFNγ production assay results for the M1085T sample.

[0315] Figure 26: Depicted on Figure 8 Raw data of IL-2 ELISA assay results.

[0316] Fig. 27 : Presented in Fig. 9 and 10 raw data of metabolic matrix and metabolite analysis results.

[0317] Fig.28 : Presented in Fig.11 The raw data of the relative telomere length analysis results.

[0318] Fig.29 : Presented in Fig.12 and 15 Raw data of unique CD3 sequences and clonal diversity analysis results.

[0319] Fig.30 : Shows a comparison between various 2nd generation (2A process) and 3.1 generation process implementations.

[0320] Fig.31 : The table describes various features of implementation schemes for Generation 2, Generation 2.1, and Generation 3.0 processes.

[0321] Fig.32 : Overview of media conditions for an embodiment of the 3rd generation process (referred to as generation 3.1).

[0322] Fig.33 : The table describes various features of implementation schemes for Generation 2, Generation 2.1, and Generation 3.0 processes.

[0323] Fig.34 : The table compares various features of implementation schemes of Generation 2 and Generation 3.0 processes.

[0324] Fig.35 : The table provides the culture medium usage for various embodiments of the expansion process.

[0325] Fig.36 : Phenotypic Comparison: Passage 3.0 and Passage 3.1 Process Embodiments Show Comparable CD28, CD27 and CD57 Expression. Passage 3.1 testing (which included the addition of OKT-3 and feeder cells on day 0) reached maximum capacity of the culture flask at the time of harvest.

[0326] Fig.37: The 3rd generation final product has higher IFNγ production. IFNγ analysis (by ELISA) was assessed in the culture frozen supernatant to compare the two processes. Fresh TIL products of each 2nd generation (e.g., day 22) and 3rd generation process (e.g., day 16) were used to stimulate each tumor overnight with coated anti-CD3 plates. Each bar represented here refers to the IFNγ level of stimulated, unstimulated and culture medium control.

[0327] Fig.38 : A) Unique CDR3 sequence analysis of TIL final products: Columns show 1×10 collected from passage 2 (e.g., day 22) and passage 3 (e.g., days 14 to 16). 6 The number of unique TCR B clone types identified by each cell. Based on the number of unique peptide CDRs in the sample, the 3rd generation showed higher clonal diversity compared to the 2nd generation. B) Diversity index of TIL final products: The Shanon entropy diversity index is a more reliable and common comparative measure. The 3rd generation showed slightly higher diversity compared to the 2nd generation. C) Unique CDR3 sequence analysis of the 3rd generation, 3.1st generation control and 3.1st generation test process TIL final products of L4063 and L4064. The column shows 1x10 collected from the 16th day of the 3rd generation and 3.1st generation process harvest. 6 1 generation. The number of unique TCR B clone types identified by each cell. Based on the number of unique peptide CDRs in the sample, the 3.1 generation shows slightly higher clonal diversity compared to the 3 generation. D) Diversity index of the TIL final product of L4063 and L4064 under the 3 generation, 3.1 generation control and 3.1 generation test process. The Shannon entropy diversity index is a more reliable and common comparative measure. L4063 and L4064 of the 3.1 generation condition show slightly higher diversity compared to the 3 generation process.

[0328] Fig.39 : 199 sequences were shared between the final products of the 3rd and 2nd generations, corresponding to the top 80% of 97.07% coming from unique CDR3 sequences shared by the final products of the 2nd and 3rd generations.

[0329] Fig.40 : 1833 sequences were shared between the final products of the 3rd and 2nd generations, corresponding to the top 80% of 99.45% coming from unique CDR3 sequences shared by the final products of the 2nd and 3rd generations.

[0330] Fig.41 : Schematic representation of an exemplary embodiment of a 3rd generation process (16 day process).

[0331] Fig.42: A schematic diagram of an exemplary embodiment of amplifying TILs from hematopoietic malignancies using a 3rd generation process. On day 0, T cell fractions (CD3+, CD45+) are isolated from lymphocyte-rich apheresis products, whole blood, or (fresh or thawed) tumor digests using positive or negative selection methods, i.e., T cell markers (CD2, CD3, etc. or other cells are removed to leave T cells) or gradient centrifugation to remove T cells.

[0332] Fig.43 : Schematic representation of an exemplary embodiment of a 3rd generation process (16 day process).

[0333] Fig.44 : Provides a process overview of an exemplary embodiment of a Generation 3.1 process (16-day process) (Generation 3.1 testing).

[0334] Fig.45 : Provide data for TIL proliferation, average total viable cell count per tumor fragment, viability percentage on harvest day, and total viable cell count (TVC) on harvest day for an exemplary embodiment of a 3rd generation process (3.0, 3.1 control, 3.1 test). The 3.1 generation test (which includes the addition of OKT-3 and feeder cells on day 0) reaches the maximum capacity of the culture bottle at the time of harvest. If a maximum of 4 culture bottles are started on day 0, each TVC harvest should be multiplied by 4.

[0335] Fig.46 : Bar graph depicts total viable cell count (TVC) and percent viability for an exemplary embodiment of a 16 day process for a 3 generation process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test).

[0336] Fig.47 : The data provided show that an exemplary embodiment of the 3 passage process (passage 3.0, 3.1 control, and 3.1 test) produces cells that show comparable expression of CD28, CD27, and CD57.

[0337] Fig.48:The data provided show that the TIL memory state between the cells produced by the exemplary embodiments of the 3rd generation process (generation 3.0, generation 3.1 control and generation 3.1 test) is comparable. The memory state of REP TIL is depicted as follows: CD4+ or CD8+ TIL memory subsets are divided into different memory subsets. Initial (CD45RA+CD62L+), CM: central memory (CD45RA-CD62L+), EM: effector memory (CD45RA-CD62L-), TEMRA / TEFF: RA+ effector memory / effector cells (CD45RA+CD62L+). The bar graph presented is the positive percentage of CD45+ / -CD62L+ / - when gated on CD4+ or CD8+.

[0338] Fig.49 : The data provided show that cells generated by the exemplary embodiment of the 3rd generation process (generation 3.0, generation 3.1 control and generation 3.1 test) have comparable TIL activation / exhaustion markers when gated on CD4+. Activation and exhaustion of REP TILs were determined by multicolor flow cytometry. The harvested TIL samples were stained with flow cytometry antibodies (CD3-BUV395, PD-1-BV421, 2B4 / CD244-PB, CD8-BB515, CD25-BUV563, BTLA-PE, KLRG1-PE-Dazzle594, TIM-3-BV650, CD194 / CCR4-APC, CD4-VioGreen, TIGIT-PerCP-eFluor 710, CD183-BV711, CD69-APC-R700, CD95-BUV737, CD127-PE-Cy7, CD103-BV786, LAG-3-APC-eFluor780). The bar graphs presented are the CD4+ or CD8+ TIL percentages of REP TIL.

[0339] Fig.50: The data provided show that cells generated by the exemplary embodiment of the 3rd generation process (generation 3.0, generation 3.0, generation 3.1 control and generation 3.1) have comparable TIL activation / exhaustion markers when gated on CD8+. Activation and exhaustion of REPTIL were determined by multicolor flow cytometry. The harvested TIL samples were stained with flow cytometry antibodies (CD3-BUV395, PD-1-BV421, 2B4 / CD244-PB, CD8-BB515, CD25-BUV563, BTLA-PE, KLRG1-PE-Dazzle594, TIM-3-BV650, CD194 / CCR4-APC, CD4-VioGreen, TIGIT-PerCP-eFluor 710, CD183-BV711, CD69-APC-R700, CD95-BUV737, CD127-PE-Cy7, CD103-BV786, LAG-3-APC-eFluor780). The bar graphs presented are the CD4+ or CD8+ TIL percentages of REP TIL.

[0340] Fig.51 :The data provided show that the final product of generation 3.1 exhibits higher IFN-γ production. IFNγ analysis ELISA was evaluated in culture frozen supernatant to compare the two processes. Fresh TIL products of each tumor at each harvest day were used to stimulate overnight with coated anti-CD3 plates. Each bar represented here refers to the level of IFNγ stimulated, unstimulated and culture medium control.

[0341] Fig.52 : Data presented show that supernatant IL-2 concentrations are comparable between exemplary embodiments of the 3rd generation process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard media. Left: L4063 - Generation 2 Standard Media. Right: L4064 - CTS Optimizer Media. *ELISA was performed using AIM V diluent

[0342] Fig.53 : The data provided show that the supernatant metabolite concentrations between the exemplary embodiments of the 3rd generation process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test) are comparable. L4063 TILs were expanded in standard medium. L4064 TILs were expanded in CTS Optimizer medium.

[0343] Fig.54: Telomere length analysis of exemplary embodiments of the Generation 3 process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test). Telomere length analysis of cells generated by tumor identifiers L4063 and L4064: Relative telomere length (RTL) values ​​indicate the average telomere fluorescence per chromosome / genome in cells generated by the Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test processes using the DAKO kit relative to the telomere fluorescence per chromosome / genome in the control cell line (1301 leukemia cell line).

[0344] Fig.55 : Schematic diagram of an exemplary embodiment of the Generation 3.1 Testing (Generation 3.1 Optimization) process (16 to 17 day process).

[0345] Fig.56 : Schematic representation of an exemplary embodiment of a 3rd generation process (16 day process).

[0346] Figures 57A-57B : Comparative table of exemplary 2nd generation and exemplary 3rd generation processes with exemplary differences highlighted.

[0347] Fig.58 : Schematic representation of an exemplary embodiment of the preparation timeline for a 3rd generation process (16 / 17 day process).

[0348] Fig.59 : Schematic representation of an exemplary embodiment of a 3rd generation process (14 to 16 day process).

[0349] Fig.60 : Summary of data on day 16 / 17 for three engineered runs of an exemplary Generation 3 process implementation.

[0350] Fig.61 : Data on TIL extension phenotype: Shown are TIL identification (ID)-defined differentiation characteristics of cells generated from two engineering runs of an exemplary 3rd generation process embodiment.

[0351] Fig.62 : Data on the extended phenotype of TILs expanded from lung tumors: Shown are TIL identification (ID)-defined differentiation signatures of cells generated from two method development (PD) runs of an exemplary 3rd generation process implementation using lung tumor tissue.

[0352] Fig.63 : Data on the extended phenotype (purity, discrimination and memory) of TILs expanded from ovarian tumors: Shown are the purity, discrimination and memory phenotype characteristics of cells expanded from ovarian tumors using exemplary passage 2, passage 3.1 and FR ER (frozen tumor, early REP) process embodiments; * Indicates an untested condition;Y Indicates sampling problems, low TVC counts, or non-viable cells upon thaw.

[0353] Fig.64 : Shown are data characterizing the gating strategy for TILs (gating strata shown) and extended phenotypic characterization of cells generated from two engineering runs of an exemplary 3rd generation process embodiment.

[0354] Fig.65 : Shown is the gating strategy for identifying TILs (gating strata shown) and data on extended phenotypic characterization of the CD4+ and CD8+ subsets of cells generated by two engineering runs of an exemplary 3rd generation process embodiment.

[0355] Fig.66 : Shown are data for granulolytic enzyme B ELISA analysis of cells produced from two engineered runs of an exemplary 3rd generation process embodiment.

[0356] Figures 67A-67B : Schematic representation of an exemplary embodiment of a 3rd generation process (16 day process).

[0357] Fig.68 : Schematic representation of an exemplary embodiment of a 3rd generation process (16 day process).

[0358] Fig.69 : Comparison of implementation schemes of 2nd, 2.1st and 3rd generation processes (16 day process).

[0359] Fig.70 : Comparison of implementation schemes of 2nd, 2.1st and 3rd generation processes (16 day process).

[0360] Fig.71 : Components of the 3rd generation implementation scheme.

[0361] Fig.72 : Comparison of flow charts of 3rd generation implementation schemes (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test).

[0362] Fig.73 : Presents total viable cell counts and fold expansions for exemplary Generation 3 embodiments (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and serum-free cell culture medium.

[0363] Fig.74 : Presents the % viability fraction upon reactivation, culture scale-up, and TIL harvest for exemplary Generation 3 embodiments (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and serum-free cell culture medium.

[0364] Fig.75 : Presented is the phenotypic characterization of the final TIL products generated by processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (3.0, 3.1 control, and 3.1 test) using standard cell culture medium and CTS serum-free cell culture medium.

[0365] Fig.76 : Presented is a memory marker analysis of TIL products generated by processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (3.0, 3.1 control, and 3.1 test) using standard cell culture medium and CTS serum-free cell culture medium.

[0366] Fig.77 : Presented are activation and exhaustion markers of TILs generated from processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (3.0, 3.1 control, and 3.1 test) using standard cell culture medium and CTS serum-free cell culture medium and subsequently subjected to CD4+ gated cell sorting.

[0367] Fig.78 : Presented are activation and exhaustion markers of TILs generated from processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (3.0, 3.1 control, and 3.1 test) using standard cell culture medium and CTS serum-free cell culture medium and subsequently subjected to CD8+ gated cell sorting.

[0368] Fig.79 : Presented are the IFN-γ production (pg / mL) fractions of the final TIL products generated by processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium.

[0369] Fig.80 : Presented is an analysis of IL-2 concentration (pg / mL) in spent media (collected at reactivation, culture scale-up, and TIL harvest) from processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium.

[0370] Fig.81 : Presented are glucose concentrations (g / L) in spent media (collected at reactivation, culture scale-up, and TIL harvest) from processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium.

[0371] Fig.82 : Presented are lactate concentrations (g / L) in spent media (collected at reactivation, culture scale-up, and TIL harvest) from processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium.

[0372] Fig.83 : Presented are glutamine concentrations (mmol / L) in spent media (collected at reactivation, culture scale-up, and TIL harvest) from processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium.

[0373] Fig.84 : Presented are glutamax concentrations (mmol / L) in spent media (collected at reactivation, culture scale-up, and TIL harvest) from processing L4063 and L4064 tumor samples in an exemplary 3rd generation process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium.

[0374] Fig.85 : Presented are ammonia concentrations (mmol / L) in spent media (collected at reactivation, culture scale-up, and TIL harvest) from processing L4063 and L4064 tumor samples in an exemplary Generation 3 process (Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium. Telomere length analysis of an exemplary embodiment of a Generation 3 process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test). Telomere length analysis of cells generated by tumor identifiers L4063 and L4064: Relative telomere length (RTL) values ​​indicate the average telomere fluorescence per chromosome / genome in cells generated by the Generation 3.0, Generation 3.1 Control, and Generation 3.1 Test processes using the DAKO kit relative to the telomere fluorescence per chromosome / genome in a control cell line (1301 leukemia cell line).

[0375] Fig.86: Telomere length analysis of TILs produced by exemplary embodiments of the 3rd generation process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test) using standard cell culture medium and CTS serum-free cell culture medium. Telomere length analysis of cells produced by tumor identifiers L4063 and L4064: Relative telomere length (RTL) values ​​indicate the average telomere fluorescence per chromosome / genome in cells produced by the 3.0, 3.1 Control, and 3.1 Test processes using the DAKO kit relative to the telomere fluorescence per chromosome / genome in the control cell line (1301 leukemia cell line).

[0376] Fig.87 : Summary of TCR Vβ repertoires of TILs produced by exemplary embodiments of the 3rd generation process (generation 3.0, generation 3.1 control, generation 3.1 test) using standard cell culture medium and CTS serum-free cell culture medium. Described are the clonality of TILs measured by TCR Vβ repertoires with unique CDR3 sequences for the final TIL products produced by the 3.0, 3.1 control and 3.1 test processes for tumor identifiers L4063 and L4064.

[0377] Fig.88 : Comparison of TILs produced by exemplary embodiments of the 3rd generation process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test) with respect to the frequency of unique CDR3 sequences in the TIL harvest products of processing L4063 tumor samples.

[0378] Fig.89 : Comparison of the percentage of unique CDR3 sequences shared in TIL harvested cell products of L4063 tumor samples processed from TILs produced by exemplary embodiments of the 3rd generation process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test): 975 sequences were shared between the Generation 3.0 and Generation 3.1 Test final products, equivalent to 88%, with the top 80% coming from unique CDR3 sequences shared between the Generation 3.0 and Generation 3.1 Test final products.

[0379] Fig.90 : Comparison of the percentage of shared unique CDR3 sequences in TIL harvested cell products from processing L4064 tumor samples produced by exemplary embodiments of the 3rd generation process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test): 2163 sequences were shared between the Generation 3.0 and Generation 3.1 Test final products, equivalent to 87%, with the top 80% coming from unique CDR3 sequences shared between the Generation 3.0 and Generation 3.1 Test final products.

[0380] Fig.91: Comparison of TILs produced by exemplary embodiments of the 3rd generation process (Generation 3.0, Generation 3.1 Control, Generation 3.1 Test) with respect to the frequency of unique CDR3 sequences in TIL harvest products processing L4064 tumor samples.

[0381] Fig.92 : Shown are components of an exemplary embodiment of a Generation 3 process (Generation 3 Optimization, 16 to 17 day process).

[0382] Fig.93 : Accept standard table.

[0383] Fig.94 : Cell count reactivation day.

[0384] Fig.95 : Cell counting scale-up day.

[0385] Fig.96 : Cell counts of harvested L4063.

[0386] Fig.97 : Cell counts of harvested L4064.

[0387] Fig.98 : Flow data.

[0388] Fig.99 : Flow data.

[0389] Fig.100 : Flow data.

[0390] Fig.101 : Flow data.

[0391] Figure 102: IFN-γ production data Figure 7-L 4063.

[0392] Figure 103: IFN-γ production data Figure 7-L 4064.

[0393] Figure 104: ELISA analysis of IL-2 concentration data.

[0394] Fig.105 : Summary table of metabolic data.

[0395] Fig.106 : Summarizes the data.

[0396] Fig.107 : Summarizes the data.

[0397] Fig.108 : Shannon diversity index.

[0398] Fig.109: The diagram of exemplary process 2A provides an overview of steps A to F.

[0399] Fig.110 : Structures IA and IB are provided, and the cylinders refer to individual polypeptide binding domains. Structures IA and IB contain three linearly connected TNFRSF binding domains derived from, for example, 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then connected to a second trivalent protein via an IgG1-Fc (including CH3 and CH2 domains), which is used to connect the two trivalent proteins together via a disulfide bond (small oblong), thereby stabilizing the structure and providing an agonist capable of bringing together the intracellular signaling domains of the six receptors with the signaling protein to form a signaling complex. The TNFRSF binding domain represented as a cylinder can be a scFv domain, which includes, for example, a VH and VL chain connected by a linker, which can include hydrophilic residues and Gly and Ser sequences that provide flexibility and Glu and Lys that provide solubility.

[0400] Fig.111 : Overview of 2nd and 3rd generation processes using biopsy samples.

[0401] Fig.112 : An exemplary embodiment of a 3rd generation process.

[0402] Brief description of sequence

[0403] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0404] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0405] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0406] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0407] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0408] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0409] SEQ ID NO:7 is the amino acid sequence of recombinant human IL-15 protein.

[0410] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0411] SEQ ID NO: 9 is the amino acid sequence of human 4-1BB.

[0412] SEQ ID NO: 10 is the amino acid sequence of murine 4-1BB.

[0413] SEQ ID NO: 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0414] SEQ ID NO: 12 is the light chain of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0415] SEQ ID NO: 13 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0416] SEQ ID NO: 14 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0417] SEQ ID NO: 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0418] SEQ ID NO: 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0419] SEQ ID NO: 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0420] SEQ ID NO: 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0421] SEQ ID NO: 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0422] SEQ ID NO: 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody Urolumab (PF-05082566).

[0423] SEQ ID NO: 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0424] SEQ ID NO: 22 is the light chain of the 4-1BB agonist monoclonal antibody Urelumab (BMS-663513).

[0425] SEQ ID NO: 23 is the heavy chain variable region (V H ).

[0426] SEQ ID NO: 24 is the light chain variable region (V L ).

[0427] SEQ ID NO: 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody usurelumab (BMS-663513).

[0428] SEQ ID NO: 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody usurelumab (BMS-663513).

[0429] SEQ ID NO: 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody usurelumab (BMS-663513).

[0430] SEQ ID NO: 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody usurelumab (BMS-663513).

[0431] SEQ ID NO: 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody usurelumab (BMS-663513).

[0432] SEQ ID NO: 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody usurelumab (BMS-663513).

[0433] SEQ ID NO:31 is the Fc domain of a TNFRSF agonist fusion protein.

[0434] SEQ ID NO:32 is the linker of the TNFRSF agonist fusion protein.

[0435] SEQ ID NO: 33 is the linker of the TNFRSF agonist fusion protein.

[0436] SEQ ID NO: 34 is the linker of the TNFRSF agonist fusion protein.

[0437] SEQ ID NO: 35 is the linker of the TNFRSF agonist fusion protein.

[0438] SEQ ID NO: 36 is the linker of the TNFRSF agonist fusion protein.

[0439] SEQ ID NO: 37 is the linker of the TNFRSF agonist fusion protein.

[0440] SEQ ID NO: 38 is the linker of the TNFRSF agonist fusion protein.

[0441] SEQ ID NO: 39 is the linker of the TNFRSF agonist fusion protein.

[0442] SEQ ID NO:40 is the linker of the TNFRSF agonist fusion protein.

[0443] SEQ ID NO:41 is the linker of the TNFRSF agonist fusion protein.

[0444] SEQ ID NO:42 is the Fc domain of a TNFRSF agonist fusion protein.

[0445] SEQ ID NO:43 is the linker of the TNFRSF agonist fusion protein.

[0446] SEQ ID NO:44 is the linker of the TNFRSF agonist fusion protein.

[0447] SEQ ID NO:45 is the linker of the TNFRSF agonist fusion protein.

[0448] SEQ ID NO:46 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0449] SEQ ID NO:47 is the soluble portion of the 4-1BBL polypeptide.

[0450] SEQ ID NO: 48 is the heavy chain variable region (V H ).

[0451] SEQ ID NO: 49 is the light chain variable region (V L ).

[0452] SEQ ID NO: 50 is the heavy chain variable region (V H ).

[0453] SEQ ID NO: 51 is the light chain variable region (V L ).

[0454] SEQ ID NO: 52 is the heavy chain variable region (V H ).

[0455] SEQ ID NO: 53 is the light chain variable region (V L ).

[0456] SEQ ID NO:54 is the amino acid sequence of human OX40.

[0457] SEQ ID NO:55 is the amino acid sequence of murine OX40.

[0458] SEQ ID NO:56 is the heavy chain of the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).

[0459] SEQ ID NO:57 is the light chain of the OX40 agonist monoclonal antibody tavoliximumab (MEDI-0562).

[0460] SEQ ID NO: 58 is the heavy chain variable region (V H ).

[0461] SEQ ID NO: 59 is the light chain variable region (V L ).

[0462] SEQ ID NO:60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tavoliximumab (MEDI-0562).

[0463] SEQ ID NO:61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tavoliximumab (MEDI-0562).

[0464] SEQ ID NO:62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tavoliximumab (MEDI-0562).

[0465] SEQ ID NO:63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tavoliximumab (MEDI-0562).

[0466] SEQ ID NO:64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tavoliximumab (MEDI-0562).

[0467] SEQ ID NO:65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tavoliximumab (MEDI-0562).

[0468] SEQ ID NO:66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0469] SEQ ID NO:67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0470] SEQ ID NO: 68 is the heavy chain variable region (V H ).

[0471] SEQ ID NO: 69 is the light chain variable region (V L ).

[0472] SEQ ID NO:70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0473] SEQ ID NO:71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0474] SEQ ID NO:72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0475] SEQ ID NO:73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0476] SEQ ID NO:74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0477] SEQ ID NO:75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0478] SEQ ID NO:76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0479] SEQ ID NO:77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0480] SEQ ID NO: 78 is the heavy chain variable region (V H ).

[0481] SEQ ID NO: 79 is the light chain variable region (V L ).

[0482] SEQ ID NO:80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0483] SEQ ID NO:81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0484] SEQ ID NO:82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0485] SEQ ID NO:83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0486] SEQ ID NO:84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0487] SEQ ID NO:85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0488] SEQ ID NO: 86 is the heavy chain variable region (V H ).

[0489] SEQ ID NO: 87 is the light chain variable region (V L ).

[0490] SEQ ID NO:88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0491] SEQ ID NO:89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0492] SEQ ID NO:90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0493] SEQ ID NO:91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0494] SEQ ID NO:92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0495] SEQ ID NO:93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0496] SEQ ID NO: 94 is the heavy chain variable region (VH ).

[0497] SEQ ID NO: 95 is the light chain variable region (V L ).

[0498] SEQ ID NO:96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0499] SEQ ID NO:97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0500] SEQ ID NO:98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0501] SEQ ID NO:99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0502] SEQ ID NO: 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0503] SEQ ID NO: 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0504] SEQ ID NO: 102 is the amino acid sequence of OX40 ligand (OX40L).

[0505] SEQ ID NO: 103 is the soluble portion of the OX40L polypeptide.

[0506] SEQ ID NO: 104 is an alternative soluble portion of the OX40L polypeptide.

[0507] SEQ ID NO: 105 is the heavy chain variable region (V H ).

[0508] SEQ ID NO: 106 is the light chain variable region (V L ).

[0509] SEQ ID NO: 107 is the heavy chain variable region (V H ).

[0510] SEQ ID NO: 108 is the light chain variable region (V L ).

[0511] SEQ ID NO: 109 is the heavy chain variable region (V H ).

[0512] SEQ ID NO: 110 is the light chain variable region (V L ).

[0513] SEQ ID NO: 111 is the heavy chain variable region (V H ).

[0514] SEQ ID NO: 112 is the light chain variable region (V L ).

[0515] SEQ ID NO: 113 is the heavy chain variable region (V H ).

[0516] SEQ ID NO: 114 is the light chain variable region (V L ).

[0517] SEQ ID NO: 115 is the heavy chain variable region (V H ).

[0518] SEQ ID NO: 116 is the light chain variable region (V L ).

[0519] SEQ ID NO: 117 is the heavy chain variable region (V H ).

[0520] SEQ ID NO: 118 is the heavy chain variable region (V H ).

[0521] SEQ ID NO: 119 is the light chain variable region (V L ).

[0522] SEQ ID NO: 120 is the light chain variable region (V L ).

[0523] SEQ ID NO: 121 is the heavy chain variable region (V H ).

[0524] SEQ ID NO: 122 is the heavy chain variable region (V H ).

[0525] SEQ ID NO: 123 is the light chain variable region (V L ).

[0526] SEQ ID NO: 124 is the light chain variable region (V L ).

[0527] SEQ ID NO: 125 is the heavy chain variable region (V H ).

[0528] SEQ ID NO: 126 is the light chain variable region (V L ). DETAILED DESCRIPTION

[0529] I. Definitions

[0530] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0531] The term "in vivo" refers to events that occur inside the body of a subject.

[0532] The term "in vitro" refers to events that occur outside the body of a subject. In vitro assays encompass cell-based assays, in which living or dead cells are employed, and can also encompass cell-free assays that do not employ intact cells.

[0533] The term "ex vivo" refers to an event involving treatment or procedure performed on cells, tissues and / or organs removed from the body of a subject. Suitably, the cells, tissues and / or organs may be returned to the subject using a surgical or therapeutic method.

[0534] The term "rapid expansion" refers to that antigen-specific TIL increases at least about 3 times (or 4, 5, 6, 7, 8 or 9 times) in quantity within a one-week period, more preferably increases at least about 10 times (or 20, 30, 40, 50, 60, 70, 80 or 90 times) within a one-week period, or most preferably increases at least about 100 times within a one-week period. Some rapid expansion schemes are summarized below.

[0535] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to a population of cells that are originally obtained as leukocytes but that leave the subject's bloodstream and migrate 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. TIL includes primary and secondary TIL. "Primary TIL" is obtained from patient tissue samples as summarized herein (sometimes referred to as "freshly obtained" or "freshly separated"), and "secondary TIL" is any TIL cell colony through amplification or hyperplasia as described herein, including but not limited to bulk TIL (bulk TIL) and amplified TIL ("REP TIL" or "TIL after REP"). TIL cell colonies may include genetically modified TIL.

[0536] In this article, "cell population" (including TIL) refers to a group of cells with common characteristics. Generally speaking, the number of cells in a population is usually between 1×10 6 Up to 1×10 10 Different TIL populations contain different numbers. For example, initial growth of primary TIL in the presence of IL-2 produces approximately 1×10 8 REP expansion is usually performed to provide 1.5 × 10 9 to 1.5×10 10 In some embodiments, REP expansion is performed to provide 2.3×10 10 –13.7×10 10 A group of individuals.

[0537] "Cryopreserved TILs" herein refer to TILs, whether primary, bulk or expanded (REP TILs), that have been processed and stored in the range of about -150°C to -60°C. Conventional methods of cryopreservation are also described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that can be used as a source of primary TILs.

[0538] "Thawed cryopreserved TILs" herein refers to a population of TILs that were previously cryopreserved and then processed to return to room temperature or higher, including but not limited to cell culture temperature or a temperature where the TILs can be administered to a patient.

[0539] TILs can generally be defined biochemically (using cell surface markers) or functionally (by their ability to infiltrate tumors and effect therapy). TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. In addition and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into a patient.

[0540] The term "cryopreservation media" or "cryopreservation medium" refers to any culture medium that can be used to cryopreserve cells. Such culture media may include culture media that contain 7% to 10% DMSO. Exemplary culture media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation medium available from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by its trade name " CS10" is mentioned. CS10 medium is a serum-free, animal component-free medium containing DMSO.

[0541] The term "central memory T cells" 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. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2 and BMI1. Central memory T cells mainly secrete IL-2 and CD40L as effector molecules after TCR priming. Central memory T cells are the main CD4 cells in the blood and are enriched in human lymph nodes and tonsils.

[0542] The term "effector memory T cells" refers to a subset of human or mammalian T cells that are CD45R0+ like central memory T cells but have lost the ability to constitutively express CCR7 (CCR7 lo ) and heterogeneous or low expression of CD62L (CD62L lo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high amounts of inflammatory cytokines, including interferon gamma, IL-4 and IL-5, after antigen stimulation. Effector memory T cells are the main blood CD8 cells and are enriched in human lungs, liver and intestines. CD8+ effector memory T cells carry a large amount of perforin.

[0543] The term "closed system" refers to a system that is isolated from the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, for example, but are not limited to, closed G containers. Once the tumor segment is added to the closed system, the system is isolated from the external environment until the TIL is ready to be administered to the patient.

[0544] The terms "fragmenting," "fragment," and "fragmented" as used herein describe the process of destroying a tumor, including mechanical fragmentation methods such as breaking, cutting, dividing, and pulverizing tumor tissue, as well as any other method for destroying the physical structure of tumor tissue.

[0545] The term "fine needle aspirate" or FNA refers to a type of biopsy procedure that can be used for sampling or diagnostic procedures, including tumor sampling, in which samples are collected but the tumor is not removed or excised. In fine needle aspiration, a hollow needle (e.g., 25 to 18 gauge) is inserted into a tumor or an area containing a tumor, and fluid and cells (including tissue) are obtained for further analysis or amplification as described herein. When performing FNA, cells are removed and the histological structure of tissue cells is not retained. FNA may include TIL. In some examples, fine needle aspiration biopsy is performed using an ultrasound-guided fine needle aspiration biopsy needle. FNA needles can be purchased from Becton Dickinson, Covidien, etc.

[0546] The term "core biopsy" or "core needle biopsy" refers to a biopsy procedure that can be used for sampling or diagnostic procedures, including tumor sampling, wherein samples are collected but not removed or removed tumors. In a core biopsy, a hollow needle (e.g., No. 16 to No. 11) is inserted into a tumor or a region containing a tumor, and fluid and cells (including tissue) are obtained for further analysis or amplification as described herein. When performing a core biopsy, cells can be removed under the histological structure of retaining some tissue cells because they have a larger needle size compared to FNA. The core biopsy needle is typically a size that can retain the tumor histological structure of at least some parts. The core biopsy object may include TIL. In some examples, a core needle biopsy is performed using a biopsy instrument, vacuum-assisted core needle biopsy instrument, stereotactic guidance core needle biopsy instrument, ultrasound guidance core needle biopsy instrument, and MRI guidance core needle biopsy instrument that can be purchased from Bard Medical, Becton Dickinson, etc.

[0547] 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 (PBMCs are antigen presenting cells), peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0548] The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBL are isolated from whole blood or apheresis products of a donor. In some embodiments, PBL are isolated from whole blood or apheresis products of a donor by positive or negative selection of T cell phenotypes (such as CD3+CD45+ T cell phenotypes).

[0549] The term "anti-CD3 antibody" refers to an antibody or variant thereof, such as a monoclonal antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes human, humanized, chimeric or murine antibodies. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0550] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or a biosimilar or variant thereof directed against the CD3 receptor in the T cell antigen receptor of mature T cells, including human, humanized, chimeric or murine antibodies, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or its variants, conservative amino acid substitutions, glycosylated forms or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). The hybridoma capable of producing OKT-3 is deposited in the American Type Culture Collection and the assigned ATCC accession number is CRL 8001. The hybridoma capable of producing OKT-3 is also deposited in the European Authentication Cell Culture Center (ECACC) and the assigned catalog number is 86022706.

[0551] Table 1. Amino acid sequence of muromonab

[0552]

[0553] The term "IL-2" (also referred to herein as "IL2") refers to a T cell growth factor called interleukin-2, and includes all forms of IL-2, including its human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars and variants. IL-2 is described in, for example, 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 a recombinant human IL-2 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available from multiple suppliers, 22 million IU per single-use vial), and recombinant IL-2 forms supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-209-b) and other commercially available equivalents from other suppliers. Aldesleukin (desalanyl-1, human IL-2 substituted with serine-125) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses IL-2 in a PEGylated form as described herein, including PEGylated IL2 prodrug NKTR-214 (available from Nektar Therapeutics, South San Francisco, CA, USA). NKTR-214 and PEGylated IL-2 suitable for the present invention are described in U.S. Patent Application Publication No. US 2014 / 0328791A1 and International Patent Application Publication No. WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4902,502, the disclosures of which are incorporated herein by reference. IL-2 preparations suitable for the present invention are described in U.S. Patent No. 6,706,289, the disclosures of which are incorporated herein by reference.

[0554] Table 2. Amino acid sequences of interleukins

[0555]

[0556] The term "IL-4" (also referred to herein as "IL4") refers to a cytokine called interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the initial Helper T cells (Th0 cells) differentiate into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Under the activation of 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 B cell type switching to express IgE and IgG 1 Recombinant human IL-4 suitable for use in the present invention can be purchased from a number of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (Human IL-15 Recombinant Protein, Catalog No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 5).

[0557] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine called interleukin 7, which can be obtained from stromal cells 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 (a heterodimer composed of the IL-7 receptor α and the common γ chain receptor), which is a series of signals important for the development of T cells in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention can be purchased from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 6).

[0558] The term "IL-15" (also referred to herein as "IL15") refers to a T cell growth factor called interleukin-15, and includes all forms of IL-2, including its human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars and variants. IL-15 is described in, for example, 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 can be purchased from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (Human IL-15 Recombinant Protein, Catalog No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 7).

[0559] The term "IL-21" (also referred to herein as "IL21") refers to a pleiotropic cytokine protein called interleukin-21, and includes all forms of IL-21, including its human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars, and variants. IL-21 is described in, for example, Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosures of which are incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 + T cell production. 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 can be purchased from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for the present invention is given in Table 2 (SEQ ID NO: 8).

[0560] When an "anti-tumor effective amount", "tumor suppression 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, infection or metastasis range and patient (subject) condition differences of the subject. It is generally stated that the pharmaceutical composition comprising tumor infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein can be 10 4 Up to 10 11 cells / kg body weight (e.g. 10 5 Up to 10 6 , 10 5 Up to 10 10 , 10 5 Up to 10 11 , 10 6 Up to 10 10 , 10 6 Up to 10 11 , 10 7 Up to 10 11 , 10 7 Up to 10 10 , 10 8 Up to 10 11 , 10 8 Up to 10 10 , 10 9 Up to 10 11 or 10 9 Up to 10 10 The dosage of the invention can be administered at a dose of 100 cells / kg body weight, including all integer values ​​within those ranges. Tumor infiltrating lymphocytes (including genetically modified cytotoxic lymphocytes in some cases) compositions can also be administered multiple times at these doses. Tumor infiltrating lymphocytes (including genetically modified cytotoxic lymphocytes in some cases) can be administered using infusion techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by a technician in the medical field by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0561] The terms "hematological malignancy" or related terms refer to cancers and tumors of mammalian hematopoietic and lymphoid tissues (including but not limited to blood, bone marrow, lymph nodes, and tissues of the lymphatic system). Hematological malignancies are also called "liquid tumors". Hematological malignancies include but are not limited to acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma and non-Hodgkin's lymphoma. The term "B-cell hematological malignancy" refers to a hematological malignancy that affects B cells.

[0562] The term "solid tumor" refers to an abnormal mass of tissue, usually without cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The tissue structure of a solid tumor includes interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells in which cancer cells are dispersed and can provide a supporting microenvironment.

[0563] The term "liquid tumor" refers to an abnormal mass of cells with fluid properties. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors are also referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors (including liquid tumors circulating in peripheral blood) are also referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only in the type of tissue from which the cells are derived.

[0564] As used herein, the term "microenvironment" may refer to the entire parenchymal or hematological tumor microenvironment or may refer to individual cell subsets in the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical stimulation that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, encourage treatment resistance, and provide space for dominant metastatic growth" as described by Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, the immune system rarely clears tumors because of the immunosuppressive effects of the microenvironment.

[0565] In one embodiment, the present invention includes a method for treating cancer using a TIL colony, wherein the patient is first treated with non-myeloablative chemotherapy before TIL is infused according to the present invention. In some embodiments, a TIL colony can be provided, wherein the patient is first treated with non-myeloablative chemotherapy before TIL is infused according to the present invention. In one embodiment, non-myeloablative chemotherapy is cyclophosphamide 60mg / kg / d for 2 days (the 27th and 26th day before TIL infusion) and fludarabine 25mg / m2 / d for 5 days (the 27th to 23rd day before TIL infusion). In one embodiment, after non-myeloablative chemotherapy and TIL infusion (the 0th day) according to the present invention, the patient receives intravenous infusion of 720,000IU / kg intravenous IL-2 every 8 hours to physiological tolerance.

[0566] Experimental findings indicate that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing for elements of the immune system ("cytokine sinks"). Therefore, some embodiments of the present invention perform a lymphocyte depletion step (sometimes also referred to as "immunosuppressive conditioning") on the patient prior to introduction of the rTILs of the present invention.

[0567] As used herein, the terms "co-administered", "co-administered", "administered in combination with", "simultaneously" and "concurrently" encompass the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, for example, a combination of at least one potassium channel agonist and a plurality of TILs) to a subject so that the two active pharmaceutical ingredients and / or their metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of separate compositions and administration of a composition in which two agents are present are preferred.

[0568] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds as described herein that is sufficient to achieve the intended application, including but not limited to disease treatment. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the subject and disease condition (e.g., the subject's weight, age, and sex) being treated, the severity of the disease condition, or the mode of administration. The term also applies to doses that will induce a specific response in the target cell (e.g., reduction in platelet adhesion and / or cell migration). The specific dose will vary depending on the specific compound selected, the dosing regimen followed, whether the compound is administered in combination with other compounds, the time point of administration, the tissue to which it is to be administered, and the physical delivery system carrying the compound.

[0569] The term "treatment / treating / treat" and similar terms refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, that is, completely or partially preventing a disease or its symptoms, and / or the effect may be therapeutic, that is, partially or completely curing a disease and / or the adverse effects of a disease. As used herein, "treatment" encompasses any treatment of a mammal (particularly a human) disease, and includes: (a) preventing the disease from occurring in a subject, who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, that is, stopping the development or progression of the disease; and (c) alleviating the disease, that is, causing the disease to regress and / or alleviating one or more symptoms of the disease. "Treatment" also encompasses the delivery of an agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition that can induce an immune response or confer immunity in the absence of a disease condition, such as in the case of a vaccine.

[0570] The term "heterologous" when used to refer to portions of a nucleic acid or protein means that the nucleic acid or protein comprises two or more subsequences that do not have the same relationship to each other in nature. For example, the nucleic acid is generally recombinantly produced and has two or more sequences from unrelated genes that are arranged to make a new functional nucleic acid, such as a promoter from one source and a coding region from another source, or the coding regions are from different sources. Similarly, a heterologous protein means that the protein comprises two or more subsequences that do not have the same relationship to each other in nature (e.g., a fusion protein).

[0571] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their semantic terms, e.g., "99% identical"), when referring to two or more nucleic acids or polypeptides, mean that the two or more sequences or subsequences are identical or have a specified percentage of nucleotides or amino acid residues that are identical when compared and aligned (introducing gaps where necessary) for maximum correspondence and not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software are known in the art and can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs that can determine percent sequence identity include, for example, the BLAST package available from the BLAST website of the National Center for Biotechnology Information of the U.S. government. Comparisons between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign (available from DNASTAR) are other publicly available software programs for aligning sequences. One skilled in the art can determine the appropriate parameters for a particular alignment software to maximize alignment. In certain embodiments, the default parameters of the alignment software are used.

[0572] As used herein, the term "variant" encompasses, but is not limited to, proteins, antibodies or fusion proteins comprising an amino acid sequence different from the amino acid sequence of a reference protein, antibody or fusion protein, the difference being that there are 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. Compared to the amino acid sequence of the reference antibody, the amino acid sequence of the variant may contain one or more conservative substitutions. Conservative substitutions may involve, for example, substitutions of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody, protein or fusion protein. The term variant also includes pegylated antibodies or proteins.

[0573] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to a population of cells that are originally obtained as leukocytes but that leave the subject's bloodstream and migrate 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 primary and secondary TILs. "Primary TILs" are those obtained from patient tissue samples (sometimes referred to as "freshly obtained" or "freshly isolated") as outlined herein, and "secondary TILs" are any TIL cell populations that have been expanded or proliferated as described herein, including but not limited to bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs may include, for example, a second expanded TIL or a second additional expanded TIL (such as, for example, those described in step D of FIG. 1, including TILs referred to as reREP TILs).

[0574] TILs can generally be defined biochemically (using cell surface markers) or functionally (by their ability to infiltrate tumors and achieve treatment). TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. In addition and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into the patient. TILs can be further characterized by efficacy - for example, if, for example, interferon (IFN) release is greater than about 50pg / mL, greater than about 100pg / mL, greater than about 150pg / mL, or greater than about 200pg / mL, then TILs can be considered effective. For example, TILs can be considered effective if the interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, greater than about 1000 pg / mL.

[0575] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients (such as other drugs) may also be incorporated into the described compositions and methods.

[0576] The term "about" or "approximately" refers to within the statistically significant range of a numerical value. Such a range may be within the order of magnitude of a given numerical value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, and even more preferably within 5%. The permissible deviations encompassed by the term "about" or "approximately" depend on the particular research system and may be readily understood by those skilled in the art. In addition, the terms "about" and "approximately" as used herein mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and features are not exact and do not need to be exact, but may be approximate and / or larger or smaller values ​​as required, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or features are "about" or "approximately", whether or not so clearly stated. It has been noted that embodiments of very different sizes, shapes, and dimensions may adopt the described arrangements.

[0577] When the transition terms "comprising," "consisting essentially of," and "consisting of," are used in the appended claims in their original and amended forms, they define the scope of the claims with respect to which uncited additional claim elements or steps, if any, 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, uncited elements, methods, steps, or materials. The term "consisting of" excludes any elements, steps, or materials not specified in the claim, and in the latter case excludes impurities normally associated with the specified materials. The term "consisting essentially of" limits the scope of the claim to the specified elements, steps, or materials and those that do not materially affect the basic and novel characteristics of the claimed invention. All compositions, methods, and kits embodying the present invention described herein may be more specifically defined in alternative embodiments by any of the transition terms "comprising," "consisting essentially of," and "consisting of."

[0578] II. TIL Manufacturing Methods (Embodiment of a Generation 3 Process, Optionally Including Defined Media)

[0579] Without being bound by any particular theory, it is believed that the priming first expansion of activation of T cells as described in the methods of the present invention, followed by a rapid second expansion to boost the activation of T cells, allows the preparation of expanded T cells that retain a "younger" phenotype, and it is therefore expected that the expanded T cells of the present invention can exhibit higher cytotoxicity against cancer cells than T cells expanded by other methods. In particular, it is believed that priming the activation of T cells by exposure to anti-CD3 antibodies (e.g., OKT-3), IL-2, and optionally antigen presenting cells (APCs) as taught by the methods of the present invention and then boosting by subsequent exposure to additional anti-CD-3 antibodies (e.g., OKT-3), IL-2, and APCs, limits or avoids the maturation of T cells in culture, thereby generating a population of T cells with a less mature phenotype that is less exhausted by culture expansion and exhibits higher cytotoxicity against cancer cells. In some embodiments, the step of rapid second expansion is divided into the following steps to achieve culture scaling up: (a) rapid second expansion is performed by culturing T cells in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for about 3 to 4 days, followed by (b) transferring the T cells in the small-scale culture to a second container (e.g., a G-REX 500MCS container) that is larger than the first container and culturing the T cells from the small-scale culture in a larger-scale culture in the second container for about 4 to 7 days. In some embodiments, the rapid expansion step is divided into the following steps to achieve horizontal expansion of the culture scale: (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container (e.g., a G-REX 100MCS container) for about 3 to 4 days, followed by (b) transferring and distributing 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 containers of equal size to the first container, wherein in each second container, the portion of T cells from the first small-scale culture transferred to the second container is cultured in the second small-scale culture for about 4 to 7 days. In some embodiments, the rapid expansion step is divided into the following multiple steps to achieve horizontal expansion and scale-up of the culture scale: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for about 3 to 4 days, followed by (b) transferring and distributing T cells from the 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 containers (e.g., a G-REX 500MCS container) that are larger than the first container, wherein in each second container, the portion of T cells from the small-scale culture transferred to the 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 the following multiple steps to achieve horizontal expansion and scale-up of the culture scale: (a) rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for about 4 days, followed by (b) transferring and distributing T cells from the small-scale culture to 2, 3 or 4 second containers (e.g., a G-REX 500MCS container) that are larger than the first container, wherein in each second container, the portion of T cells from the small-scale culture transferred to the second container is cultured in the larger-scale culture for about 5 days.

[0580] In some embodiments, the rapid second expansion is performed after T cell activation achieved by the priming first expansion begins to decrease, slow, wane, or subside.

[0581] In some embodiments, the rapid second expansion is performed after T cell activation achieved by the priming first expansion has been reduced by or about 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%, %, 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% after.

[0582] In some embodiments, the rapid second expansion is performed after T cell activation achieved by the priming first expansion has been reduced to a percentage in the range of at or about 1% to 100%.

[0583] In some embodiments, the rapid second expansion is performed after T cell activation achieved by the priming first expansion has been reduced by a percentage in the range of or about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0584] In some embodiments, the rapid second expansion is performed after T cell activation achieved by the priming first expansion has been reduced by at least or about 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%, %, 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% after.

[0585] In some embodiments, the rapid second expansion is performed after T cell activation achieved by the priming first expansion has been reduced by at most about or by about 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%, %, 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% after.

[0586] In some embodiments, the reduction in T cell activation achieved by the priming first expansion is determined by a reduction in the amount of interferon gamma released by the T cells in response to antigenic stimulation.

[0587] In some embodiments, the primed first expansion of T cells is performed over a period of up to or about 7 days or about 8 days.

[0588] In some embodiments, the primed first expansion of T cells is performed over a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

[0589] In some embodiments, the primed first expansion of T cells is performed over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

[0590] In some embodiments, the rapid second expansion of T cells is performed over a period of up to or about 11 days.

[0591] In some embodiments, the rapid second expansion of T cells is performed over a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0592] In some embodiments, the rapid second expansion of T cells is performed over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0593] In some embodiments, the primed first expansion of T cells is performed over a period of from at or about 1 day to at or about 7 days and the rapid second expansion of T cells is performed over a period of from at or about 1 day to at or about 11 days.

[0594] In some embodiments, the primed first expansion of T cells is performed over a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days and the rapid second expansion of T cells is performed over a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or about 11 days.

[0595] In some embodiments, the primed first expansion of T cells is performed over a period of from at or about 1 day to at or about 8 days and the rapid second expansion of T cells is performed over a period of from at or about 1 day to at or about 9 days.

[0596] In some embodiments, the primed first expansion of T cells is performed over a period of 8 days and the rapid second expansion of T cells is performed over a period of 9 days.

[0597] In some embodiments, the primed first expansion of T cells is performed over a period of from at or about 1 day to at or about 7 days and the rapid second expansion of T cells is performed over a period of from at or about 1 day to at or about 9 days.

[0598] In some embodiments, the primed first expansion of T cells is performed over a period of 7 days and the rapid second expansion of T cells is performed over a period of 9 days.

[0599] In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs).

[0600] In some embodiments, the T cells are bone marrow infiltrating lymphocytes (MIL).

[0601] In some embodiments, the T cells are peripheral blood lymphocytes (PBL).

[0602] In some embodiments, the T cells are obtained from a donor with cancer.

[0603] In some embodiments, the T cells are obtained from TILs of a tumor resected from a patient suffering from cancer.

[0604] In some embodiments, the T cells are obtained from MILs from the bone marrow of a patient with a hematological malignancy.

[0605] In some embodiments, T cells are obtained from PBLs of peripheral blood mononuclear cells (PBMCs) from donors. In some embodiments, donors suffer from cancer. In some embodiments, cancer is a cancer 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 papilloma virus, 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 papilloma virus, 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, donors suffer from tumors. 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.

[0606] In certain aspects of the invention, immune effector cells (e.g., T cells) can be collected from a unit of blood from a subject, using any number of techniques known to skilled technicians (such as FICOLL separation) to obtain. In a preferred aspect, cells from the subject's circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma portion and optionally placed in a suitable buffer or culture medium for subsequent processing steps. In one embodiment, cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution lacks calcium, and may lack magnesium or may lack many (if not all) divalent cations. In one aspect, T cells are separated from peripheral blood lymphocytes, which can be separated by, for example, centrifugation through a PERCOLL gradient or by countercurrent centrifugal elutriation to lyse red blood cells and deplete monocytes.

[0607] In some embodiments, T cells are PBLs separated from the whole blood of the donor or the apheresis product rich in lymphocytes. In some embodiments, the donor suffers from cancer. In some embodiments, the cancer is a cancer 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 papilloma virus, 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 papilloma virus, 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 suffers from 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 suffers from a hematological malignancy. In some embodiments, PBLs are isolated from whole blood or apheresis products enriched for lymphocytes using positive or negative selection methods, i.e., using markers of T cell phenotype such as CD3+CD45+ to remove PBLs, or removing non-T cell phenotype cells and leaving PBLs. In other embodiments, PBLs are isolated by gradient centrifugation. After isolating PBLs from donor tissue, the priming first expansion of PBLs can be performed according to the priming first expansion step in any of the methods described herein, by a suitable number of isolated PBLs (in some embodiments, about 1×10 7PBL) were inoculated into the priming first expansion culture to initiate.

[0608] An exemplary TIL process called Process 3 (also referred to herein as Generation 3) containing some of these features is depicted in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ), and some of the advantages of this embodiment of the present invention over process 2A are described in Figures 1, 2, 30 and 31 (particularly, for example Figure 1B and / or Figure 1C ). Two embodiments of process 3 are shown in Figures 1 and 30 (particularly, for example Figure 1B and / or Figure 1C ). Process 2A or Generation 2 is also described in U.S. Patent Publication No. 2018 / .0280436, which is incorporated herein by reference in its entirety.

[0609] As discussed herein and generally outlined, TILs are taken from patient samples and operated using a TIL expansion process described herein and referred to as the 3rd generation to expand their number before being transplanted into a patient. In some embodiments, TILs may optionally be subjected to genetic manipulation as discussed below. In some embodiments, TILs may be cryopreserved before or after amplification. Once thawed, they may also be restimulated to increase their metabolism before being infused into a patient.

[0610] In some embodiments, as discussed in detail below and in the Examples and Figures, the priming first amplification (including the process referred to herein as rapid pre-amplification (pre-REP) and FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) step B) is shortened to 1 to 8 days and the rapid second amplification (including the process referred to herein as the rapid amplification protocol (REP) and FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) step D) is shortened to 1 to 9 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the priming first amplification (including the process referred to herein as rapid pre-amplification (pre-REP) and FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) step B) is shortened to 1 to 8 days and the rapid second amplification (including the process referred to herein as the rapid amplification protocol (REP) and FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) step D) is shortened to 1 to 8 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the priming first amplification (including the process referred to herein as rapid amplification pre-(pre-REP) and Figure 1 (particularly, for example Figure 1B and / or Figure 1C) step B) is shortened to 1 to 7 days and the rapid second amplification (including the process referred to herein as the rapid amplification protocol (REP) and FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) step D) is shortened to 1 to 9 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the priming first amplification (including the process referred to herein as rapid pre-amplification (pre-REP) and FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The process shown in step B) is 1 to 7 days and the rapid second amplification (including the process referred to herein as the rapid amplification protocol (REP) and Figure 1 (especially for example Figure 1B and / or Figure 1C ) The process shown in step D) is 1 to 10 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) step B described amplification) is shortened to 8 days and a rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 7 to 9 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is 8 days and the rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 8 to 9 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is shortened to 7 days and the rapid second amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step D) is 7 to 8 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) step B described amplification) is shortened to 8 days and a rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 8 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is 8 days and the rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 9 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is 8 days and the rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 10 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is 7 days and the rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 7 to 10 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is 7 days and the rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 8 to 10 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is 7 days and the rapid second amplification (eg, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification described in step D) is 9 to 10 days. In some embodiments, the priming first amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step B) is shortened to 7 days and the rapid second amplification (e.g., FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) The amplification described in step D) is 7 to 9 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the priming first amplification is combined with a rapid second amplification (e.g., FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The combination of step B and step D) is 14 to 16 days. In particular, it is contemplated that certain embodiments of the invention comprise a priming first expansion step, wherein the TILs are activated by exposure to an anti-CD3 antibody (e.g., OKT-3) in the presence of IL-2 or by 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 priming first expansion step as described above are a first TIL population, i.e., a primary cell population.

[0611] The following "steps" are coded A, B, C, etc. with reference to FIG. 1 (particularly, for example Figure 1B and / or Figure 1C) and with reference to certain non-limiting embodiments described herein. Figure 1B and / or Figure 1C ) are exemplary and any combination or order of steps, as well as additional steps, repeated steps and / or omission of steps are contemplated in the present application and the methods disclosed herein.

[0612] A. Step A: Obtaining Patient Tumor Samples

[0613] Generally, TILs are initially obtained from patient tumor samples ("primary TILs") or circulating lymphocytes (such as peripheral blood lymphocytes, including peripheral blood lymphocytes with TIL-like characteristics), and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

[0614] Patient tumor samples can be obtained using methods known in the art, usually via surgical resection, needle biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells. In general, tumor samples can be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. Tumor samples can also be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be any cancer type, 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, cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (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 TIL is obtained from malignant melanoma tumors, because reports indicate that these tumors have particularly high levels of TIL.

[0615] Once obtained, tumor samples are typically fragmented using sharp instruments into pieces ranging from 1 to about 8 mm. 3 Small pieces between 2 and 3 mm 3 Particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. The tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2mM glutamate, 10mcg / mL gentamicin, 30 units / mL of DNase, and 1.0mg / mL of collagenase), followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in an enzymatic medium and mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 Incubate for 30 minutes, then repeat mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue pieces are present and produced. At the end of this process, if the cell suspension contains a large amount of red blood cells or dead cells, FICOLL branched hydrophilic polysaccharides can be used for density gradient separation to remove these cells. Alternative methods known in the art can be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, and its disclosure is incorporated herein by reference. Any of the aforementioned methods can be used in the method for amplifying TIL in any embodiment described herein or in the method for treating cancer.

[0616] As indicated above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumors are not fragmented. In some embodiments, the solid tumors are not fragmented and are enzymatically digested as whole tumors. In some embodiments, the tumors are digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase. In some embodiments, the tumors are digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1 to 2 hours. In some embodiments, the tumors are digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase at 37°C, 5% CO 2 In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and hyaluronidase at 37°C, 5% CO 2 In some embodiments, the tumors are digested overnight under constant rotation. In some embodiments, the tumors are incubated at 37°C, 5% CO 2 In some embodiments, whole tumors are combined with enzymes to form a tumor digestion reaction mixture.

[0617] In some embodiments, the tumor is reconstituted with freeze-dried enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0618] In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is a 100 mg / ml 10X working stock solution.

[0619] In some embodiments, the enzyme mixture comprises DNase.In some embodiments, the working stock solution of DNase is a 10,000 IU / ml 10X working stock solution.

[0620] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is a 10-mg / ml 10X working stock solution.

[0621] In some embodiments, the enzyme cocktail comprises 10 mg / ml collagenase, 1000 IU / ml DNase, and 1 mg / ml hyaluronidase.

[0622] In some embodiments, the enzyme cocktail comprises 10 mg / ml collagenase, 500 IU / ml DNase, and 1 mg / ml hyaluronidase.

[0623] In general, the cell suspension obtained from the tumor is referred to as a "primary cell population" or a "freshly obtained" or "freshly isolated" cell population. In certain embodiments, the freshly obtained TIL cell population is exposed to a cell culture medium comprising antigen presenting cells, IL-12 and OKT-3.

[0624] In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is segmentation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatically catalyzed tumor digests and tumor fragments obtained from a patient. In one embodiment, TILs may initially be cultured from enzymatically catalyzed tumor digests and tumor fragments obtained from a patient.

[0625] In some embodiments, when the tumor is a solid tumor, in, for example (as shown in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) After obtaining the tumor sample in step A) provided in ), the tumor is physically fragmented. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor and no cryopreservation is performed. 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 than 40 fragments or pieces are placed in each container for priming first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for priming first amplification. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for priming first amplification. In some embodiments, the plurality of fragments includes about 4 to about 50 fragments, each of which has about 27 mm 3 In some embodiments, the plurality of segments comprises about 30 to about 60 segments having a total volume of about 1300 mm 3 To about 1500mm 3 In some embodiments, the plurality of segments comprises about 50 segments having a total volume of about 1350 mm 3In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0626] In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharp segmentation. In some embodiments, tumor fragments are between about 1 mm 3 With 10mm 3 In some embodiments, the tumor fragment is between about 1 mm 3 With 8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor segment is about 6 mm 3 In some embodiments, the tumor segment is about 7 mm 3 In some embodiments, the tumor segment is about 8 mm 3 In some embodiments, the tumor segment 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 x 1-4 mm x 1-4 mm. In some embodiments, the tumor fragment is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor fragment is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor fragment is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor fragment is 4 mm x 4 mm x 4 mm.

[0627] In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic, necrotic and / or adipose tissue on each slice. In some embodiments, the tumor is fragmented to minimize the amount of hemorrhagic tissue on each slice. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each slice. In some embodiments, the tumor is fragmented to minimize the amount of adipose tissue on each slice. In certain embodiments, the step of fragmenting the tumor is an in vitro or ex vivo method.

[0628] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without using a scalpel to perform a sawing action. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are produced by incubation in an enzyme culture medium such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10mg / mL gentamicin, 30U / mL DNase, and 1.0mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in an enzyme culture medium, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C in 5% CO 2 The cells were incubated for 30 minutes and then mechanically disrupted again for approximately 1 minute. The cells were incubated at 37°C in 5% CO 2 After incubation for another 30 minutes in 5% CO, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large pieces of tissue remain after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, either again at 37°C in 5% CO. 2 Incubate for 30 minutes in 4% paraformaldehyde. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells.

[0629] In some embodiments, the cell suspension prior to the priming first expansion step is referred to as the "primary cell population" or a "freshly obtained" or "freshly isolated" cell population.

[0630] In some embodiments, cells can be optionally frozen after sample isolation (e.g., after obtaining a tumor sample and / or after obtaining a cell suspension from a tumor sample) and stored frozen prior to entering the expansion described in step B, which is described in further detail below and in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ).

[0631] 1. Core / small biopsy-derived TILs

[0632] In some embodiments, TILs are initially obtained from patient tumor samples by core biopsy or similar procedures ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, and optionally assessed for phenotypic and metabolic parameters.

[0633] In some embodiments, patient tumor samples can be obtained using methods known in the art, usually via small biopsy, core biopsy, needle biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells. In general, tumor samples can be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. Tumor samples can also be liquid tumors, such as tumors obtained from hematological malignancies. In some embodiments, samples can be from multiple small tumor samples or biopsies. In some embodiments, samples can include multiple tumor samples from a single tumor of the same patient. In some embodiments, samples can include multiple tumor samples from one, two, three or four tumors of the same patient. In some embodiments, samples can include multiple tumor samples from multiple tumors of the same patient. Solid tumors can be any cancer type, 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 (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (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, as these tumors are reported to have particularly high levels of TILs.

[0634] In general, cell suspensions obtained from tumor cores or fragments are referred to as "primary cell populations" or "freshly obtained" new or "freshly isolated" cell populations. In certain embodiments, freshly obtained TIL cell populations are exposed to a cell culture medium comprising antigen presenting cells, IL-2, and OKT-3.

[0635] In some embodiments, if the tumor is metastatic and the primary lesion has been effectively treated / removed in the past, it may be necessary to remove a metastatic lesion. In some embodiments, the least invasive approach is to remove available skin lesions or cervical or axillary lymph nodes. In some embodiments, skin lesions are removed or a small biopsy thereof is removed. In some embodiments, lymph nodes or a small biopsy thereof are removed. In some embodiments, lung or liver metastatic lesions or intra-abdominal or thoracic lymph nodes or a small biopsy thereof may be used.

[0636] In some embodiments, the tumor is a melanoma. In some embodiments, the small biopsy of a melanoma comprises a mole or a portion thereof.

[0637] In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin around a suspicious mole. In some embodiments, the punch biopsy is obtained by pressing a circular blade into the skin and removing a circular piece of skin. In some embodiments, the small biopsy is a punch biopsy and removes a circular portion of the tumor.

[0638] In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and removes the entire mole or growth. In some embodiments, the small biopsy is an excisional biopsy and removes the entire mole or growth along with a small edge of normal-looking skin.

[0639] In some embodiments, the small biopsy is an incisional biopsy. In some embodiments, the small biopsy is an incisional biopsy and only the mole or growth of the most irregular part is collected. In some embodiments, the small biopsy is an incisional biopsy and the incisional biopsy is used when other techniques cannot be completed, such as when the suspicious mole is very large.

[0640] In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Typically, bronchoscopy is under anesthesia of the patient, a small tool is passed through the nose or mouth, down to the throat and into the bronchial passage, wherein the small tool is used to remove some tissues. In some embodiments, wherein the tumor or growth cannot be reached via bronchoscopy, a transthoracic needle aspiration biopsy can be used. Typically, transthoracic needle aspiration biopsy is also under anesthesia of the patient, a needle is inserted directly into a suspicious site through the skin to remove the tissue of a small sample. In some embodiments, transthoracic needle aspiration biopsy may require interventional radiology (e.g., using x-rays or CT scans to guide the needle). In some embodiments, a small biopsy is obtained by needle aspiration biopsy. In some embodiments, a small biopsy is obtained through endoscopic ultrasound (e.g., an endoscope with a lamp is placed in the esophagus through the mouth). In some embodiments, a small biopsy is obtained through surgery.

[0641] In some embodiments, the small biopsy is a head and neck biopsy. In some embodiments, the small biopsy is an incisional biopsy. In some embodiments, the small biopsy is an incisional biopsy, wherein a small piece of tissue is removed from the abnormal appearance area. In some embodiments, if the abnormal area is easily accessible, sample collection may not require hospitalization. In some embodiments, if the tumor is deeper in the mouth or throat, the biopsy may need to be performed under general anesthesia in the operating room. In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy, wherein the entire area is removed. In some embodiments, the small biopsy is a fine needle aspiration (FNA). In some embodiments, the small biopsy is a fine needle aspiration (FNA), wherein a very fine needle attached to a syringe is used to extract (suction) cells from a tumor or mass. In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the small biopsy is a punch biopsy, wherein a piece of suspicious part is removed using punching forceps.

[0642] In some embodiments, the small biopsy is a cervical biopsy. In some embodiments, the small biopsy is obtained via a colposcope. Typically, the colposcopic method uses a magnifying instrument (colposcope) with a light attached to a binocular magnifier, which is then used to biopsy a small portion of the cervical surface. In some embodiments, the small biopsy is a cervical cone excision / cone biopsy. In some embodiments, the small biopsy is a cervical cone excision / cone biopsy, where outpatient surgery may be required to remove a larger piece of cervical tissue. In some embodiments, the cone biopsy can be used as an initial treatment in addition to helping confirm the diagnosis.

[0643] The term "solid tumor" refers to an abnormal mass of tissue, usually without cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas and lymphomas, such as lung cancer, breast cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer and bladder cancer. In some embodiments, 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 solid tumors includes interdependent tissue compartments, including parenchyma (cancer cells) and cancer cells dispersed therein and supporting stromal cells that can provide a supportive microenvironment.

[0644] In some embodiments, the sample from the tumor is obtained as a fine needle aspirate (FNA), a core biopsy, a small biopsy (including, for example, a punch biopsy). In some embodiments, the sample is first placed in the G-Rex 10. In some embodiments, when there are 1 or 2 core biopsy and / or small biopsy samples, the sample is first placed in the G-Rex 10. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more core biopsy and / or small biopsy samples, the sample is first placed in the G-Rex 100. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more core biopsy and / or small biopsy samples, the sample is first placed in the G-Rex 500.

[0645] The FNA may be obtained from a tumor selected from the group consisting of lung tumors, melanomas, head and neck tumors, cervical tumors, ovarian tumors, pancreatic tumors, glioblastomas, colorectal tumors, and sarcomas. In some embodiments, the FNA is obtained from a lung tumor, such as a lung tumor from a non-small cell lung cancer (NSCLC) patient. In some cases, the NSCLC patient has previously undergone surgical treatment.

[0646] TIL as described herein can be obtained from FNA sample.In some cases, FNA sample is to obtain or separate from patient using the fine gauge needle of the range from 18 gauge needle to 25 gauge needle.The fine gauge needle can be 18, 19, 20, 21, 22, 23, 24 or 25.In some embodiments, the FNA sample from patient can contain at least 400,000 TIL, for example 400,000 TIL, 450,000 TIL, 500,000 TIL, 550,000 TIL, 600,000 TIL, 650,000 TIL, 700,000 TIL, 750,000 TIL, 800,000 TIL, 850,000 TIL, 900,000 TIL, 950,000 TIL or more.

[0647] In some cases, TIL as described herein is obtained from core biopsy sample.In some cases, core biopsy sample is to obtain or separate from patient using the surgical or medical needle of the scope from 11 gauge needle to 16 gauge needle.The needle can be No. 11, No. 12, No. 13, No. 14, No. 15 or No. 16.In some embodiments, core biopsy sample from patient can contain at least 400,000 TIL, for example 400,000 TIL, 450,000 TIL, 500,000 TIL, 550,000 TIL, 600,000 TIL, 650,000 TIL, 700,000 TIL, 750,000 TIL, 800,000 TIL, 850,000 TIL, 900,000 TIL, 950,000 TIL or more.

[0648] Generally, the harvested cell suspension is referred to as the "primary cell population" or "freshly harvested" cell population.

[0649] In some embodiments, the TILs are not obtained from a tumor digest.In some embodiments, the solid tumor core is not fragmented.

[0650] In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are produced by incubation in an enzyme culture medium such as, but not limited to, RPMI1640, 2mM GlutaMAX, 10mg / mL gentamicin, 30U / mL DNase, and 1.0mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme culture medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be incubated at 37°C in 5% CO 2 The cells were incubated for 30 minutes and then mechanically disrupted again for approximately 1 minute. The cells were incubated at 37°C in 5% CO 2 After incubation for another 30 minutes in 5% CO, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large pieces of tissue remain after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, either again at 37°C in 5% CO. 2 Incubate for 30 minutes in 4% paraformaldehyde. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells.

[0651] 2. Method for expanding peripheral blood lymphocytes (PBL) from peripheral blood

[0652] PBL Method 1In one embodiment of the invention, PBLs are expanded using the process described herein. In one embodiment of the invention, the method comprises obtaining a PBMC sample from whole blood. In one embodiment, the method comprises enriching T cells by using negative selection of non-CD19+ fractions to separate pure T cells from PBMCs. In one embodiment, the method comprises enriching T cells by using magnetic bead-based negative selection of non-CD19+ fractions to separate pure T cells from PBMCs.

[0653] In one embodiment of the present invention, PBL method 1 is performed as follows: On day 0, cryopreserved PBMC samples are thawed and PBMCs are counted. T cells are isolated using a human pan T cell isolation kit and LS columns (Miltenyi Biotec).

[0654] PBL Method 2. In one embodiment of the invention, PBLs are expanded using PBL method 2, which comprises obtaining a PBMC sample from whole blood. T cells from PBMCs are enriched by incubating the PBMCs at 37°C for at least three hours and then isolating non-adherent cells.

[0655] In one embodiment of the present invention, PBL method 2 is performed as follows: On day 0, cryopreserved PMBC samples are thawed and PBMC cells are seeded in 6-well plates at 6 million cells per well in CM-2 medium and incubated for 3 hours at 37° C. After 3 hours, non-adherent cells (which are PBLs) are removed and counted.

[0656] PBL Method 3 In one embodiment of the invention, PBLs are expanded using PBL method 3, which comprises 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.

[0657] In one embodiment of the present invention, PBL method 3 is performed as follows: On day 0, cryopreserved PBMCs derived from peripheral blood are thawed and counted. CD19+ B cells are sorted using the CD19 Multisort Human Kit (Miltenyi Biotec). In the non-CD19+ cell fraction, T cells are purified using a human pan T cell isolation kit and LS columns (Miltenyi Biotec).

[0658] In one embodiment, PBMC is separated from a whole blood sample. In one embodiment, a PBMC sample is used as the starting material for amplifying PBL. In one embodiment, the sample is cryopreserved before the amplification process. In another embodiment, a fresh sample is used as the starting material for amplifying PBL. In one embodiment of the invention, T cells are separated from PBMC using methods known in the art. In one embodiment, T cells are separated using a human pan-T cell separation kit and LS columns. In one embodiment of the invention, T cells are separated from PBMC using an antibody selection method known in the art (e.g., CD19 negative selection).

[0659] In one embodiment of the invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify non-adherent cells. In one embodiment of the invention, the incubation time is about 3 hours. In one embodiment of the invention, the temperature is about 37° C. The non-adherent cells are then expanded using the above process.

[0660] In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising 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 1 year or more. In another embodiment, the PBMC is derived from a patient who is currently receiving an ITK inhibitor regimen such as ibrutinib.

[0661] In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor and is refractory to treatment with a kinase inhibitor or ITK inhibitor, such as ibrutinib.

[0662] In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor but is no longer receiving treatment with a kinase inhibitor or ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient who has been pretreated with a regimen comprising a kinase inhibitor or ITK inhibitor but is no longer receiving treatment with a kinase inhibitor or ITK inhibitor and has not received treatment 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 more. In another embodiment, the PBMC is derived from a patient who was previously exposed to an ITK inhibitor but has not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

[0663] In one embodiment of the invention, at day 0, cells are selected and thus sorted via CD19+. In one embodiment of the invention, selection is performed using antibody-bound beads. In one embodiment of the invention, pure T cells are isolated from PBMCs at day 0.

[0664] In one embodiment of the invention, for patients not pretreated with ibrutinib or other ITK inhibitors, 10 to 15 ml of buffy coat will produce approximately 5×10 9 PBMCs, which in turn will generate approximately 5.5 × 10 7 PBLs.

[0665] In one embodiment of the invention, for patients pre-treated with ibrutinib or other ITK inhibitors, the expansion process will generate approximately 20×10 9 In one embodiment of the present invention, 40.3×10 6 PBMCs will generate approximately 4.7 × 10 5 PBLs.

[0666] In any of the foregoing embodiments, the PBMCs may be derived from a whole blood sample, by apheresis, from a buffy coat, or from any other method known in the art for obtaining PBMCs.

[0667] 3. Method for expanding bone marrow infiltrating lymphocytes (MIL) from bone marrow-derived PBMCs

[0668] MIL Method 3 In one embodiment of the invention, the method comprises obtaining a PBMC sample from bone marrow. On day 0, PBMCs are selected and sorted by CD3+ / CD33+ / CD20+ / CD14+, and the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated and a portion of the sonicated cell fraction is added back to the selected cell fraction.

[0669] In one embodiment of the present invention, MIL method 3 is performed as follows: On day 0, a cryopreserved sample of PBMC is thawed and the PBMC is counted. The cells are stained with CD3, CD33, CD20 and CD14 antibodies and sorted using an S3e cell sorter (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+).

[0670] In one embodiment of the invention, PBMCs are obtained from bone marrow. In one embodiment, PBMCs are obtained from bone marrow via apheresis, aspiration, fine needle biopsy, or other similar means known in the art. In one embodiment, PBMCs are fresh. In another embodiment, PBMCs are cryopreserved.

[0671] In one embodiment of the invention, the MIL is expanded from 10 to 50 ml of a bone marrow aspirate. In one embodiment of the invention, 10 ml of a bone marrow aspirate is obtained from a patient. In another embodiment, 20 ml of a bone marrow aspirate is obtained from a patient. In another embodiment, 30 ml of a bone marrow aspirate is obtained from a patient. In another embodiment, 40 ml of a bone marrow aspirate is obtained from a patient. In another embodiment, 50 ml of a bone marrow aspirate is obtained from a patient.

[0672] In one embodiment of the invention, the number of PBMCs generated from about 10 to 50 ml of bone marrow aspirate is about 5×10 7 to about 10×10 7 In another embodiment, the number of PBMCs produced is about 7×10 7 PBMCs.

[0673] In one embodiment of the present invention, about 5×10 7 to about 10×10 7 PBMCs generate approximately 0.5×10 6 About 1.5×10 6 In one embodiment of the present invention, about 1×10 6 A MIL.

[0674] In one embodiment of the present invention, 12×10 6 PBMCs yield approximately 1.4 × 10 5 A MIL.

[0675] In any of the foregoing embodiments, PBMCs may be derived from a whole blood sample, bone marrow, by apheresis, from buffy coats, or from any other method known in the art for obtaining PBMCs.

[0676] B. Step B: Priming First Amplification

[0677] In some embodiments, the present methods provide younger TILs that may provide additional therapeutic benefits compared to older TILs (ie, TILs that have further replicated more times prior to administration to a subject / patient). Characteristics of young TILs have been described in the literature, e.g., Donia, et al., Scandinavian Journal of Immunology, 75:157–167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258–267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother 32:415–423 (2009); Robbins et al., J Immunol 2004;173:7125-7130; Shen et al., J Immunother, 30:123–129 (2007); Zhou et al., J Immunol 2006;174:112–115 (2008); Immunother, 28:53-62 (2005); and Tran, et al., J Immunother, 31:742-751 (2008), each of which is incorporated herein by reference.

[0678] In, for example, FIG. 1 (particularly, Figure 1B and / or Figure 1C ) After the tumor fragments and / or tumor fragments are divided or digested as described in step A, the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen presenting feeder cells) under conditions that are favorable to TILs but unfavorable to the growth of tumors and other cells. In some embodiments, IL-2, OKT-3, and feeder cells are added at the beginning of the culture along with the tumor digest and / or tumor fragments (e.g., on day 0). In some embodiments, the tumor digest and / or tumor fragments are incubated in a container at up to 60 fragments per container and 6000 IU / mL of IL-2. In some embodiments, this primary cell population is cultured for a period of several days (usually 1 to 8 days) to produce typically about 1×10 8 In some embodiments, this primary cell population is cultured for a period of several days (usually 1 to 7 days) to produce typically about 1×10 8 In some embodiments, the priming first expansion occurs for 1 to 8 days, producing typically about 1×10 8In some embodiments, the priming first expansion occurs for 1 to 7 days, producing typically about 1×10 8 In some embodiments, this priming first expansion occurs over 5 to 8 days, producing typically about 1×10 8 In some embodiments, this priming first expansion occurs over 5 to 7 days, producing typically about 1×10 8 In some embodiments, this priming first expansion occurs for about 6 to 8 days, producing typically about 1×10 8 In some embodiments, this priming first expansion occurs for about 6 to 7 days, producing typically about 1×10 8 In some embodiments, this priming first expansion occurs for about 7 to 8 days, producing typically about 1×10 8 In some embodiments, this priming first expansion occurs for about 7 days, producing typically about 1×10 8 In some embodiments, this priming first expansion occurs for about 8 days, producing typically about 1×10 8 The autologous TIL population of individual autologous TIL cells.

[0679] In a preferred embodiment, expansion of TILs may be performed using a priming first expansion step as described below and herein (e.g., such as those of FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) described in step B, which may include a process called pre-REP or priming REP and which contains feeder cells from day 0 and / or from the start of culture), followed by a rapid second expansion as described below D and described herein (step D, including a process called a rapid expansion protocol (REP) step), followed by optional cryopreservation, and then a second step D as described below and herein (including a process called a restimulation REP step). TILs obtained from this process may be optionally identified with phenotypic characteristics and metabolic parameters as described herein. In some embodiments, the tumor fragment is between about 1 mm 3 With 10mm 3 between.

[0680] In some embodiments, the first expansion medium is referred to as "CM" (abbreviation for culture medium). In some embodiments, the CM of step B consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin.

[0681] In some embodiments, there are less than or equal to 240 tumor fragments. In some embodiments, there are less than or equal to 240 tumor fragments placed in less than or equal to 4 containers. In some embodiments, the container is a GREX100MCS culture bottle. In some embodiments, less than or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each container includes less than or equal to 500mL of culture medium per container. In some embodiments, the culture medium contains IL-2. In some embodiments, the culture medium contains 6000IU / mL of IL-2. In some embodiments, the culture medium contains antigen presenting feeder cells (also referred to herein as "antigen presenting cells"). In some embodiments, the culture medium contains 2.5×10 8 antigen presenting feeder cells. In some embodiments, the culture medium comprises OKT-3. In some embodiments, the culture medium comprises 30 ng / mL of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium comprises 6000 IU / mL of IL-2, 30 ng of OKT-3, and 2.5×10 8 In some embodiments, the culture medium comprises 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen presenting feeder cells.

[0682] After the tumor fragments are prepared, the resulting cells (i.e., fragments and primary cell populations) are cultured in a medium containing IL-2, antigen presenting feeder cells, and OKT-3 under conditions that favor TIL but are unfavorable for tumor and other cell growth and that allow TIL priming and accelerated growth from the start of culture on day 0. In some embodiments, tumor digests 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 a period of several days (usually 1 to 8 days) to produce typically about 1×10 8 In some embodiments, the growth medium during the priming first expansion comprises IL-2 or a variant thereof and antigen presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured for a period of several days (usually 1 to 7 days) to produce typically about 1×10 8In some embodiments, the growth medium during the priming first expansion comprises IL-2 or a variant thereof and antigen presenting feeder cells and OKT-3. In some embodiments, the IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has 20-30×10 6 IU / mg specific activity. In some embodiments, a 1 mg vial of IL-2 stock solution has 20×10 6 IU / mg specific activity. In some embodiments, a 1 mg vial of IL-2 stock solution has 25×10 6 IU / mg specific activity. In some embodiments, a 1 mg vial of IL-2 stock solution has 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution has a specific activity of 4-8×10 6 IU / mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7×10 6 IU / mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×10 6IU / mg of IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the priming first amplification medium comprises about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6000 IU / mL of IL-2, or about 5,000 IU / mL of IL-2. In some embodiments, the priming first amplification medium comprises about 9,000 IU / mL of IL-2 to about 5,000 IU / mL of IL-2. In some embodiments, the priming first amplification medium comprises about 8,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the priming first amplification medium comprises about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the priming first expansion medium comprises about 6,000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the priming first expansion cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the priming first expansion cell culture medium further comprises IL-2. In a preferred embodiment, the priming first expansion cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the priming first expansion cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In one embodiment, the priming first expansion cell culture medium comprises between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, or about 8000 IU / mL of IL-2.

[0683] In some embodiments, the priming first expansion 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 priming first expansion medium comprises about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the priming first expansion medium comprises about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the priming first expansion medium comprises about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the priming first expansion medium comprises about 200 IU / mL of IL-15. In some embodiments, the priming first expansion cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the priming first expansion cell culture medium further comprises IL-15. In a preferred embodiment, the priming first expansion cell culture medium comprises about 180 IU / mL of IL-15.

[0684] In some embodiments, the priming first expansion 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 priming first expansion medium comprises about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the priming first expansion medium comprises about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the priming first expansion medium comprises about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the priming first expansion medium comprises about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the priming first expansion medium comprises about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the priming first expansion medium comprises about 2 IU / mL of IL-21. In some embodiments, the priming first expansion cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the priming first expansion cell culture medium comprises about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the priming first expansion cell culture medium comprises about 1 IU / mL of IL-21.

[0685] In one embodiment, the priming first expansion cell culture medium comprises OKT-3 antibody. In some embodiments, the priming first expansion cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In one embodiment, the priming first expansion cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL and about 1 μg / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises between 15 ng / ml and 30 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises 30 ng / mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0686] Table 3: Amino acid sequence of muromonab (an exemplary OKT-3 antibody)

[0687]

[0688] In some embodiments, the priming first expansion cell culture medium includes one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist includes 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, Utolumab, EU-101, fusion proteins and their fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration between 0.1 μg / mL and 100 μg / mL in the cell culture medium. In some embodiments, the added concentration of the TNFRSF agonist is sufficient to achieve a concentration between 20 μg / mL and 40 μg / mL in the cell culture medium.

[0689] In some embodiments, in addition to one or more TNFRSF agonists, the priming first expansion cell culture medium further comprises an initial concentration of about 3000 IU / mL of IL-2 and an initial concentration of about 30 ng / mL of OKT-3 antibody, and wherein the one or more TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the priming first expansion cell culture medium further comprises an initial concentration of about 6000 IU / mL of IL-2 and an initial concentration of about 30 ng / mL of OKT-3 antibody, and wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0690] In some embodiments, the priming first expansion medium is referred to as "CM" (abbreviation of 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, 25mM Hepes and 10mg / mL gentamicin. In some embodiments, CM is CM1 described in the examples (see Example A). In some embodiments, the priming first expansion occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the priming first expansion medium or the initial cell culture medium or the first cell culture medium comprises IL-2, OKT-3 and antigen presenting feeder cells (also referred to herein as feeder cells).

[0691] In some embodiments, the culture medium used in the expansion process disclosed herein is a serum-free culture medium or a defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and / or a serum replacement. In some embodiments, the serum-free or defined culture medium is used to prevent and / or reduce experimental variation due in part to batch variation of culture medium containing serum.

[0692] In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell culture medium includes but is not limited to 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 TMT cell expansion Xeno-free medium, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

[0693] In some embodiments, serum supplements or serum replacements include, but are not limited to, one or more CTS TM OpTmizer T cell expansion serum supplement, CTS TM Immune cell serum replacement, 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 antibiotics and one or more trace elements. In some embodiments, the defined 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 portion containing trace elements 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+ In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate and / or 2-mercaptoethanol.

[0694] In some embodiments, CTS TM OpTmizer TM T cell immune cell serum replacement is used with conventional growth medium, including but not limited to CTS TM OpTmizer TM T cell expansion basal medium, CTSTM OpTmizer TM T cell expansion SFM, CTS TM AIM-V medium, CST TM AIM-V SFM, LymphoONE TM T cell expansion Xeno-free medium, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

[0695] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined 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 defined medium volume. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

[0696] In some embodiments, the serum-free or defined medium is CTS TM OpTmizer TM T cell expansion SFM (ThermoFisher Scientific). Any CTS TM OpTmizer TM Preparations can be used in the present invention. TM OpTmizer TM T cell expansion SFM is 1L CTS TM OpTmizer TM T cell expansion basal medium and 26 mL CTS TM OpTmizer TM The combination of T cell expansion supplements is mixed together prior to use. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS TM OpTmizerTM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM The final concentration of immune cell serum replacement (SR) (ThermoFisher Scientific) and 2-mercaptoethanol in the culture medium was 55 μM.

[0697] In some embodiments, the defined medium is CTS TM OpTmizer TM T cell expansion SFM (ThermoFisher Scientific). Any CTS TM OpTmizer TM Preparations can be used in the present invention. TM OpTmizer TM T cell expansion SFM is 1L CTS TM OpTmizer TM T cell expansion basal medium and 26 mL CTS TM OpTmizer TM The combination of T cell expansion supplements is mixed together prior to use. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TMImmune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and also contains about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and also contains about 3000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and also contains about 6000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains about 3000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TMImmune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and also comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and also contains about 3000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and also contains about 6000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM The final concentration of immune cell serum replacement (SR) (ThermoFisher Scientific) and 2-mercaptoethanol in the culture medium was 55 μM.

[0698] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., glutamine) 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 defined medium is supplemented with glutamine at a concentration of about 2 mM (i.e. ).

[0699] In some embodiments, serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM or about 65mM. In some embodiments, serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the culture medium is 55 μM.

[0700] In some embodiments, the defined medium described in International PCT Patent Publication No. WO / 1998 / 030679 (which is incorporated herein by reference) can be used for the present invention. In the publication, serum-free eukaryotic cell culture medium is described. Serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement that can support cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises the following or is obtained by combining one or more components selected from the following 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. In some embodiments, the defined medium also comprises L-glutamine, sodium bicarbonate and / or beta-mercaptoethanol. In some embodiments, the defined medium comprises albumin or albumin substitutes and one or more components selected from the following 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 defined 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 portion containing trace elements 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+In some embodiments, the basal cell culture medium is selected from the group consisting of: Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium and Iskoff's modified Dulbecco's medium.

[0701] In some embodiments, the concentration of glycine in the defined medium is in the range of 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, and the concentration of L-threonine is about 10 to 500 mg / L. g / L, L-tryptophan is at a concentration of about 2 to 110 mg / L, L-tyrosine is at a concentration of about 3 to 175 mg / L, L-valine is at a concentration of about 5 to 500 mg / L, thiamine is at a concentration of about 1 to 20 mg / L, reduced glutathione is at a concentration of about 1 to 20 mg / L, L-ascorbic acid-2-phosphate is at a concentration of about 1 to 200 mg / L, iron-saturated transferrin is at a concentration of about 1 to 50 mg / L, insulin is at a concentration of about 1 to 100 mg / L, sodium selenite is at a concentration of about 0.000001 to 0.0001 mg / L, and albumin (e.g. I) is at a concentration of about 5000 to 50,000 mg / L.

[0702] In some embodiments, the non-trace element portion components in the defined medium are present in the concentration ranges listed in the column entitled "Concentration Ranges in 1X Medium" in Table A below. In other embodiments, the non-trace element portion components in the defined medium are present in the final concentrations listed in the column entitled "Preferred Embodiments of 1X Medium" in Table A below. In other embodiments, the defined medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element portion components of the types and concentrations listed in the column entitled "Preferred Embodiments of Supplements" in Table A below.

[0703] Table A: Concentration of some components other than trace elements

[0704]

[0705] In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L or about 2.2 g / L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-mercaptoethanol (final concentration of about 100 μM).

[0706] In some embodiments, the defined 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) can be used in the present invention. In brief, RPMI or CTS TM OpTmizer TM Used as basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS TM Immune cell serum replacement.

[0707] In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container is unfiltered. Using unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container lacks beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0708] In some embodiments, the priming first amplification (including, for example, those in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The process described in step B, which may include what is sometimes referred to as pre-REP or priming REP) is 1 to 8 days, as discussed in the Examples and Figures. In some embodiments, the priming first amplification (including such as, for example, those in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 2 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 3 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 4 C) The process described in step B, which may include what is sometimes referred to as pre-REP or priming REP) is 4 to 8 days, as discussed in the Examples and Figures. In some embodiments, the priming first amplification (including such as those in Figure 1 (particularly, for example) Figure 1B and / or Figure 1C ) The process described in step B, which may include what is sometimes referred to as pre-REP or priming REP) is 1 to 7 days, as discussed in the Examples and Figures. In some embodiments, the priming first amplification (including such as, for example, those in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 2 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 2 to 7 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 3 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 3 to 7 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 4 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 4 to 7 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 5 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) The process described in step B, which may include what is sometimes referred to as pre-REP or priming REP) is 5 to 7 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 6 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) step B, which may include what is sometimes referred to as pre-REP or priming REP) is 6 to 7 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) The process provided in step B, which may include what is sometimes referred to as pre-REP or priming REP) is 7 to 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) The process provided in step B, which may include what is sometimes referred to as pre-REP or priming REP) is 8 days. In some embodiments, the priming first amplification (including such as those in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) The process provided in step B, which may include what is sometimes referred to as a pre-REP or priming REP) is 7 days.

[0709] In some embodiments, the first TIL amplification of the priming may be carried out for 1 to 8 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the first TIL amplification of the priming may be carried out for 1 to 7 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the first TIL amplification of the priming may be carried out for 2 to 8 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the first TIL amplification of the priming may be carried out for 2 to 7 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the first TIL amplification of the priming may be carried out for 3 to 8 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the first TIL amplification of the priming may be carried out for 3 to 7 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the first TIL amplification of the priming may be carried out for 4 to 8 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the first TIL amplification of the initiation can be carried out for 4 to 7 days after fragmentation occurs and / or after the first initiation amplification step is initiated. In some embodiments, the first TIL amplification of the initiation can be carried out for 5 to 8 days after fragmentation occurs and / or after the first initiation amplification step is initiated. In some embodiments, the first TIL amplification of the initiation can be carried out for 5 to 7 days after fragmentation occurs and / or after the first initiation amplification step is initiated. In some embodiments, the first TIL amplification of the initiation can be carried out for 6 to 8 days after fragmentation occurs and / or after the first initiation amplification step is initiated. In some embodiments, the first TIL amplification of the initiation can be carried out for 6 to 7 days after fragmentation occurs and / or after the first initiation amplification step is initiated. In some embodiments, the first TIL amplification of the initiation can be carried out for 7 to 8 days after fragmentation occurs and / or after the first initiation amplification step is initiated. In some embodiments, the first TIL amplification of the initiation can be carried out for 8 days after fragmentation occurs and / or after the first initiation amplification step is initiated. In some embodiments, the priming first TIL expansion can be performed for 7 days after fragmentation occurs and / or after the first priming expansion step is initiated.

[0710] In some embodiments, the priming first expansion of TIL may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or 8 days. In some embodiments, the first TIL expansion may be performed for 1 day to 8 days. In some embodiments, the first TIL expansion may be performed for 1 day to 7 days. In some embodiments, the first TIL expansion may be performed for 2 days to 8 days. In some embodiments, the first TIL expansion may be performed for 2 days to 7 days. In some embodiments, the first TIL expansion may be performed for 3 days to 8 days. In some embodiments, the first TIL expansion may be performed for 3 days to 7 days. In some embodiments, the first TIL expansion may be performed for 4 days to 8 days. In some embodiments, the first TIL expansion may be performed for 4 days to 7 days. In some embodiments, the first TIL expansion may be performed for 5 days to 8 days. In some embodiments, the first TIL expansion may be performed for 5 days to 7 days. In some embodiments, the first TIL expansion may be performed for 6 days to 8 days. In some embodiments, the first TIL expansion may be performed for 6 days to 7 days. In some embodiments, the first TIL expansion may be performed for 7 to 8 days. In some embodiments, the first TIL expansion can be performed for 8 days. In some embodiments, the first TIL expansion can be performed for 7 days.

[0711] In some embodiments, a combination of IL-2, IL-7, IL-15 and / or IL-21 is used as a combination during the priming first expansion. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21 and any combination thereof may be included during the priming first expansion, including, for example, during the priming first expansion according to FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) during step B and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the priming first expansion. In some embodiments, IL-2, IL-15, and IL-21 and any combination thereof may be included in the process according to FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) during step B process and as described herein.

[0712] In some embodiments, the priming first amplification (e.g., according to FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C) is carried out in a closed system bioreactor. In some embodiments, a closed system is used to expand TILs as described herein. In some embodiments, a bioreactor is used. In some embodiments, a 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.

[0713] 1. Feeder cells and antigen presenting cells

[0714] In one embodiment, the priming first amplification procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added during the priming first expansion. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, for example) Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time between days 4 and 8 during the priming first expansion. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time between days 4 and 7 during the priming first expansion. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time between days 5 and 8 during the priming first expansion. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time between days 5 and 7 during the priming first expansion. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time between days 6 and 8 during the priming first expansion. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time between days 6 and 7 during the priming first expansion. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time during the priming first expansion period, during day 7 or 8. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time during the priming first expansion period up to day 7. In one embodiment, the priming first expansion procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) The expansion described in step B and those referred to as pre-REP or priming REP) do not require feeder cells (also referred to herein as "antigen presenting cells") at the start of TIL expansion, but are added at any time during the priming first expansion period during day 8.

[0715] In one embodiment, the priming first amplification procedures described herein (e.g., including those described in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C) The expansion described in step B and 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 priming first expansion. In many embodiments, feeder cells are obtained from peripheral blood mononuclear cells (PBMCs) of standard whole blood units from allogeneic healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In some embodiments, 2.5×10 8 In some embodiments, 2.5×10 feeder cells are used per vessel during the initial first expansion. 8 In some embodiments, 2.5×10 feeder cells per GREX-10 are used during the initial first expansion. 8 In some embodiments, 2.5×10 feeder cells per GREX-100 are used during the initial first expansion. 8 Feeder cells.

[0716] In general, allogeneic PBMCs are deactivated via irradiation or heat treatment and used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the replication inability of irradiated allogeneic PBMCs.

[0717] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells placed into culture on day 0 of the priming first expansion, the PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedures described herein.

[0718] In some embodiments, if the total number of viable cells on day 7 of culture in the presence of OKT3 and IL-2 does not increase from the initial number of viable cells placed in culture on day 0 of the priming first expansion, the PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 3000 IU / ml IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 6000 IU / ml IL-2.

[0719] In some embodiments, if the total number of viable cells on day 7 of culture in the presence of OKT3 and IL-2 is not increased from the initial number of viable cells placed in culture on day 0 of the first expansion of priming, the PBMC is considered to be replication incompetent and acceptable for use in the TIL expansion program described herein. In some embodiments, PBMC is cultured in the presence of 5 to 60 ng / mL OKT3 antibody and 1000 to 6000 IU / mL IL-2. In some embodiments, PBMC is cultured in the presence of 10 to 50 ng / mL OKT3 antibody and 2000 to 5000 IU / mL IL-2. In some embodiments, PBMC is cultured in the presence of 20 to 40 ng / mL OKT3 antibody and 2000 to 4000 IU / mL IL-2. In some embodiments, PBMC is cultured in the presence of 25 to 35 ng / mL OKT3 antibody and 2500 to 3500 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 6000 IU / mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng / mL OKT3 antibody and 3000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 15 ng / mL OKT3 antibody and 6000 IU / mL IL-2.

[0720] In some embodiments, antigen presenting feeder cells are PBMCs. In some embodiments, 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 amplification is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400 or about 1 to 500. In one embodiment, the ratio of TIL to antigen presenting feeder cells in the second amplification is between 1 to 50 and 1 to 300. In one embodiment, the ratio of TIL to antigen presenting feeder cells in the second amplification is between 1 to 100 and 1 to 200.

[0721] In one embodiment, the priming first amplification procedure described herein requires about 2.5×10 8 Feeder cells and about 100×10 6 In another embodiment, the priming first expansion procedure described herein requires about 2.5×10 8 Feeder cells and about 50×10 6 In yet another embodiment, the priming first expansion described herein requires about 2.5×108 Feeder cells and about 25×10 6 In yet another embodiment, the priming first expansion described herein requires about 2.5×10 8 In yet another embodiment, the priming first expansion requires one-fourth, one-third, five-twelfth, or one-half the number of feeder cells used for the rapid second expansion.

[0722] In some embodiments, the culture medium for the priming first expansion comprises IL-2. In some embodiments, the culture medium for the priming first expansion comprises 6000 IU / mL of IL-2. In some embodiments, the culture medium for the priming first expansion comprises antigen presenting feeder cells. In some embodiments, the culture medium for the priming first expansion comprises 2.5×10 8 antigen presenting feeder cells. In some embodiments, the culture medium for the priming first expansion comprises OKT-3. In some embodiments, the culture medium comprises 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture flask. In some embodiments, the culture medium comprises 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 In some embodiments, the culture medium comprises 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 In some embodiments, the culture medium comprises 2.5×10 8 In some embodiments, the culture medium comprises 500 mL of culture medium and 15 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture bottle. In some embodiments, the culture medium comprises 500 mL of culture medium and 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 In some embodiments, the culture medium comprises 500 mL of culture medium and 6000 IU / mL of IL-2, 15 μg of OKT-3, and 2.5×10 8 In some embodiments, the culture medium comprises 2.5×10 8 500 mL of culture medium and 15 μg of OKT-3 per antigen-presenting feeder cell.

[0723] In one embodiment, the priming first expansion procedure described herein requires excess feeder cells more than TIL during the second expansion period. In many embodiments, feeder cells are obtained from peripheral blood mononuclear cells (PBMC) of standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting (aAPC) cells are used instead of PBMCs.

[0724] Generally, allogeneic PBMCs are deactivated via irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the Figures and Examples.

[0725] In one embodiment, artificial antigen presenting cells are used in place of or in combination with PBMCs in the priming first expansion.

[0726] 2. Cytokines

[0727] The expansion methods described herein typically use culture medium with high doses of cytokines, particularly IL-2, as is known in the art.

[0728] Alternatively, it is possible to use a combination of cytokines to carry out the first amplification of the initiation of TIL, as generally described in International Patent Publication Nos. WO 2015 / 189356 and WO 2015 / 189357, two or more combinations of IL-2, IL-15 and IL-21, which are hereby expressly incorporated herein by reference in their entirety. Therefore, 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, wherein the latter has a specific purpose in many embodiments. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, and particularly T cells as described therein.

[0729] Table 4: Amino acid sequences of interleukins

[0730]

[0731] C. Step C: Transition from priming first amplification to rapid second amplification

[0732] In some cases, the bulk TIL population obtained from the priming first expansion (which may include what is sometimes referred to as the pre-REP expansion) includes, for example, a population of TILs obtained from, for example, FIG. 1 (particularly, for example Figure 1B and / or Figure 1C) can be subjected to a rapid second expansion (which may include an expansion sometimes referred to as a rapid expansion protocol (REP)) and then cryopreserved as discussed below. Similarly, in cases where genetically modified TILs are to be used in therapy, the expanded TIL population from the priming first expansion or the expanded TIL population from the rapid second expansion can be genetically modified for appropriate treatment prior to the expansion step or after the priming first expansion and prior to the rapid second expansion.

[0733] In some embodiments, the first amplification (eg, FIG. 1 (particularly, eg, Figure 1B and / or Figure 1C ) is stored until the phenotype is determined for selection. In some embodiments, the TILs obtained from the first priming amplification (e.g., FIG. 1 (particularly, for example Figure 1B and / or Figure 1C) The TIL of step B) shown in the figure is not stored and directly undergoes rapid second amplification. In some embodiments, the TIL obtained from the first amplification of the initiation is not cryopreserved after the first amplification of the initiation and before the rapid second amplification. In some embodiments, the conversion of the initiation first TIL amplification to the second amplification occurs after tumor fragmentation occurs and / or about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or 8 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the initiation first TIL amplification to the rapid second amplification occurs after fragmentation occurs and / or about 3 days to 7 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the initiation first TIL amplification to the rapid second amplification occurs after fragmentation occurs and / or about 3 days to 8 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the initiation first TIL amplification to the second amplification occurs after fragmentation occurs and / or about 4 days to 7 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or about 4 to 8 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or about 5 to 7 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or about 5 to 8 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or about 6 to 7 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or about 6 to 8 days after the first initiation amplification step is initiated. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or about 7 to 8 days after the first initiation amplification step is initiated. In some embodiments, the transition from the priming first TIL amplification to the second amplification occurs about 7 days after fragmentation occurs and / or after the first priming amplification step is initiated. In some embodiments, the transition from the priming first TIL amplification to the second amplification occurs about 8 days after fragmentation occurs and / or after the first priming amplification step is initiated.

[0734] In some embodiments, the conversion of the first TIL amplification of the initiation to the rapid second amplification occurs after fragmentation occurs and / or the first initiation amplification step is initiated 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or 8 days. In some embodiments, the conversion of the first TIL amplification of the initiation to the rapid second amplification occurs after fragmentation occurs and / or the first initiation amplification step is initiated 1 day to 7 days. In some embodiments, the conversion of the first TIL amplification of the initiation to the rapid second amplification occurs after fragmentation occurs and / or the first initiation amplification step is initiated 1 day to 8 days. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or the first initiation amplification step is initiated 2 days to 7 days. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or the first initiation amplification step is initiated 2 days to 8 days. In some embodiments, the conversion of the first TIL amplification of the initiation to the second amplification occurs after fragmentation occurs and / or the first initiation amplification step is initiated 3 days to 7 days. In some embodiments, the transition from the first TIL amplification of the initiation to the second amplification occurs 3 to 8 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the first TIL amplification of the initiation to the rapid second amplification occurs 4 to 7 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the first TIL amplification of the initiation to the rapid second amplification occurs 4 to 8 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the first TIL amplification of the initiation to the rapid second amplification occurs 5 to 7 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the first TIL amplification of the initiation to the rapid second amplification occurs 5 to 8 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the first TIL amplification of the initiation to the rapid second amplification occurs 6 to 7 days after fragmentation occurs and / or the first initiation amplification step is initiated. 。 In some embodiments, the transition from the initiation of the first TIL amplification to the rapid second amplification occurs 6 to 8 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the initiation of the first TIL amplification to the rapid second amplification occurs 7 to 8 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the initiation of the first TIL amplification to the rapid second amplification occurs 7 days after fragmentation occurs and / or the first initiation amplification step is initiated. In some embodiments, the transition from the initiation of the first TIL amplification to the rapid second amplification occurs 8 days after fragmentation occurs and / or the first initiation amplification step is initiated.

[0735] In some embodiments, the TILs are not stored after the primary first amplification and before the rapid second amplification, and the TILs are directly subjected to the rapid second amplification (e.g., in some embodiments, as shown in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) is not stored during the transition from step B to step D as shown in ). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, the TILs from the priming first expansion (the second TIL population) are directly subjected to a rapid second expansion without a transition period.

[0736] In some embodiments, the transition from the priming first amplification to the rapid second amplification (e.g., according to FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) is carried out in a closed system bioreactor. In some embodiments, a closed system is used to carry out TIL amplification as described herein. 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 amplification of the initiation to the second amplification of the rapid involves a scale-up of the container size. In some embodiments, the first amplification of the initiation is carried out in a smaller container than the rapid second amplification. In some embodiments, the first amplification of the initiation is carried out in a GREX-100 and the rapid second amplification is carried out in a GREX-500.

[0737] D. Step D: Rapid Second Amplification

[0738] In some embodiments, the TIL cell population is as shown in Figure 1 (particularly, for example Figure 1B and / or Figure 1C ) and a transition referred to as step C, followed by further amplification in quantity. This further amplification is referred to herein as rapid second amplification, which may include what is generally referred to in the art as a rapid amplification process (rapid amplification protocol or REP; and as shown in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) The amplification process of step D). The rapid second expansion is usually completed in a gas permeable container using a culture medium containing some components (including feeder cells, cytokine sources and anti-CD3 antibodies). In some embodiments, the TILs are transferred to a larger volume container 1 day, 2 days, 3 days or 4 days after the start of the rapid second expansion (i.e., the 8th, 9th, 10th or 11th day of the overall 3rd generation process).

[0739] In some embodiments, a rapid second expansion of TILs (which may include expansion sometimes referred to as REP; and as in FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C) The process shown in step D) can be performed using any TIL culture bottle or container known to those skilled in the art. In some embodiments, the second TIL amplification can be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification can be performed for about 1 day to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification can be performed for about 1 day to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification can be performed for about 2 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification can be performed for about 2 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification can be performed for about 3 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification can be performed for about 3 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification can be performed for about 4 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 4 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 5 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 5 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 6 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 6 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 7 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 7 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 8 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 8 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 9 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 1 day after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 2 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 3 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 4 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 5 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 6 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 7 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 8 days after the rapid second amplification is initiated.In some embodiments, the second TIL expansion can be performed about 9 days after the rapid second expansion is initiated. In some embodiments, the second TIL expansion can be performed about 10 days after the rapid second expansion is initiated.

[0740] In one embodiment, the rapid second amplification can be performed in a gas permeable container using the methods of the present disclosure (including, for example, amplification referred to as REP; and as shown in FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) is performed by the process shown in step D). In some embodiments, TILs are expanded in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen presenting cells") in a rapid second expansion. In some embodiments, TILs are expanded in the presence of IL-2, OKT-3, and feeder cells in a rapid second expansion, wherein the feeder cells are added to a final concentration that is two times, 2.4 times, 2.5 times, 3 times, 3.5 times, or 4 times the concentration of feeder cells present in the priming first expansion. For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulation may include, for example, anti-CD3 antibodies such as OKT3 at about 30 ng / ml, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (available from BioLegend, San Diego, CA, USA). TILs may be expanded to induce further TIL in vitro stimulation by including one or more cancer antigens (including their antigenic portions such as epitopes) during the second expansion period, the antigens optionally expressed from a vector in the presence of a T cell growth factor such as 300 IU / mL IL-2 or IL-15, such as human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1: 26-35 (27L) or gpl 00: 209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TILs may also be rapidly amplified by restimulation with the same cancer antigens pulsed onto HLA-A2 expressing antigen presenting cells. Alternatively, TILs may be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second amplification. In some embodiments, a second amplification occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0741] In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, or between 8000 IU / mL of IL-2.

[0742] In one embodiment, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of an OKT-3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of an OKT-3 antibody. In one embodiment, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises between 30 ng / ml and 60 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises about 60 ng / mL of OKT-3. In some embodiments, the OKT-3 antibody is muromonab.

[0743] In some embodiments, the medium for rapid second expansion comprises IL-2. In some embodiments, the medium comprises 6000 IU / mL of IL-2. In some embodiments, the medium for rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium for rapid second expansion comprises 7.5×10 8 antigen presenting feeder cells. In some embodiments, the medium for the rapid second expansion comprises OKT-3. In some embodiments, in the rapid second expansion, the medium comprises 500 mL of medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100MCS culture bottle. In some embodiments, the medium for the rapid second expansion comprises 6000 IU / mL of IL-2, 60 ng / mL of OKT-3, and 7.5×10 8 In some embodiments, the culture medium comprises 500 mL of culture medium per container and 6000 IU / mL of IL-2, 30 μg of OKT-3, and 7.5×10 8 antigen presenting feeder cells.

[0744] In some embodiments, the medium for rapid second expansion comprises IL-2. In some embodiments, the medium comprises 6000 IU / mL of IL-2. In some embodiments, the medium for rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium comprises between 5×10 8 With 7.5×10 8 In some embodiments, the medium for rapid second expansion comprises OKT-3. In some embodiments, the medium for rapid second expansion comprises 500 mL of medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS culture bottle. In some embodiments, the medium for rapid second expansion comprises 6000 IU / mL of IL-2, 60 ng / mL of OKT-3, and between 5×10 8 With 7.5×10 8 In some embodiments, the medium for rapid second expansion comprises 500 mL of medium per container and 6000 IU / mL of IL-2, 30 μg of OKT-3, and between 5×10 8 With 7.5×10 8 Antigen presenting feeder cells between the two.

[0745] 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, Utolumab, EU-101, fusion proteins and their fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration between 0.1 μg / mL and 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration between 20 μg / mL and 40 μg / mL in the cell culture medium.

[0746] In some embodiments, the cell culture medium comprises, in addition to the one or more TNFRSF agonists, IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.

[0747] 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 period. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21 and any combination thereof may be included during the second expansion period, including, for example, in accordance with FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) during step D and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as the combination during the second expansion. In some embodiments, IL-2, IL-15, and IL-21 and any combination thereof may be included in the process according to FIG. 1 (particularly, for example Figure 1B and / or Figure 1C ) during step D process and as described herein.

[0748] In some embodiments, the second expansion can be carried out 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).

[0749] In some embodiments, the second expansion 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 medium comprises about 500 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion medium comprises about 400 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion medium comprises about 300 IU / mL of IL-15 to about 100 IU / mL of IL-15. In some embodiments, the second expansion medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0750] In some embodiments, the second expansion 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 medium comprises about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion medium comprises about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion medium comprises about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion medium comprises about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion medium comprises about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the second expansion medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium also comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[0751] In some embodiments, antigen presenting feeder cell (APC) is PBMC.In one embodiment, TIL is about 1 to 10, about 1 to 15, about 1 to 20, about 1 to 25, about 1 to 30, about 1 to 35, about 1 to 40, about 1 to 45, about 1 to 50, about 1 to 75, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400 or about 1 to 500 in the ratio of PBMC in rapid expansion and / or the second expansion. In one embodiment, the ratio of TIL to PBMC in the rapid expansion and / or the second expansion is between 1-100 and 1-200.

[0752] In one embodiment, REP and / or rapid second expansion is performed in a culture flask where bulk TILs are mixed with a 100 or 200-fold excess of deactivated feeder cells, 30 ng / mL OKT3 anti-CD3 antibody, and 6000 IU / mL IL-2 in 150 ml of culture medium, where the feeder cell concentration is at least 1.1-fold (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.8X, 2X, 2.1X2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X or 4.0X of the feeder cell concentration in the priming first expansion. The medium is replaced (typically 2 / 3 of the medium is replaced by aspirating 2 / 3 of the spent medium and replacing with an equal volume of fresh medium) until the cells are transferred to the alternative growth chamber. Alternative growth chambers include G-REX culture flasks and gas permeable containers as discussed more fully below.

[0753] In some embodiments, the rapid second expansion (which may include a process called a 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.

[0754] In one embodiment, the second amplification (which may include an amplification called REP, and in FIG. 1 (particularly, e.g. Figure 1B and / or Figure 1C ) can be carried out in a 500 mL gas permeable culture bottle with a 100 cm gas permeable silicon bottom (G-Rex 100, available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5×10 6 or 10×10 6 Each TIL can be cultured with PBMC in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU of IL-2 per mL, and 30 ng of anti-CD3 (OKT3) per ml. G-Rex 100 flasks can be cultured at 37°C in 5% CO 2Incubate in GREX-100 flask. On the 5th day, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL mass can be resuspended with 150 mL of fresh culture medium containing 5% human AB serum, 6000 IU of IL-2 per mL, and added back to the original GREX-100 culture bottle. When TIL is continuously amplified in a GREX-100 culture bottle, TIL can be moved to a larger culture bottle, such as GREX-500, on the 10th or 11th day. Cells can be harvested on the 14th day of culture. Cells can be harvested on the 15th day of culture. Cells can be harvested on the 16th day of culture. In some embodiments, replacement culture medium is carried out until the cells are transferred to an alternative growth chamber. In some embodiments, 2 / 3 of the culture medium is replaced by aspirating the spent culture medium and replacing it with an equal volume of fresh culture medium. In some embodiments, an alternative growth chamber includes a GREX culture bottle and a gas permeable container as discussed more fully below.

[0755] In some embodiments, the culture medium used in the expansion process disclosed herein is a serum-free culture medium or a defined culture medium. In some embodiments, the serum-free or defined culture medium comprises a basal cell culture medium and a serum supplement and / or a serum replacement. In some embodiments, the serum-free or defined culture medium is used to prevent and / or reduce experimental variation due in part to batch variation of culture medium containing serum.

[0756] In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell culture medium includes but is not limited to 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), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

[0757] In some embodiments, serum supplements or serum replacements include, but are not limited to, one or more CTS TM OpTmizer T cell expansion serum supplement, CTSTM Immune cell serum replacement, 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 antibiotics and one or more trace elements. In some embodiments, the defined 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 portion containing trace elements 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+ In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate and / or 2-mercaptoethanol.

[0758] In some embodiments, CTS TM OpTmizer TM T cell immune cell serum replacement is used with conventional growth medium, including but not limited to CTS TM OpTmizer TM T cell expansion basal medium, CTS TM OpTmizer TM T cell expansion SFM, CTS TM AIM-V medium, CST TM AIM-V SFM, LymphoONE TMT cell expansion Xeno-free medium, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

[0759] In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined 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 defined medium volume. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

[0760] In some embodiments, the serum-free or defined medium is CTS TM OpTmizer TM T cell expansion SFM (ThermoFisher Scientific). Any CTS TM OpTmizer TM Preparations can be used in the present invention. TM OpTmizer TM T cell expansion SFM is 1L CTS TM OpTmizer TM T cell expansion basal medium and 26 mL CTS TM OpTmizer TM The combination of T cell expansion supplements is mixed together prior to use. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTSTM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM The final concentration of immune cell serum replacement (SR) (ThermoFisher Scientific) and 2-mercaptoethanol in the culture medium was 55 μM.

[0761] In some embodiments, the defined medium is CTS TM OpTmizer TM T cell expansion SFM (ThermoFisher Scientific). Any CTS TM OpTmizer TM Preparations can be used in the present invention. TM OpTmizer TM T cell expansion SFM is 1L CTS TM OpTmizer TM T cell expansion basal medium and 26 mL CTS TM OpTmizer TM The combination of T cell expansion supplements is mixed together prior to use. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and also contains about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TMImmune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and also contains about 3000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and also contains about 6000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains about 3000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and also contains about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and also comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TMImmune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and also contains about 3000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM Immune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and also contains about 6000 IU / mL of IL-2. In some embodiments, CTS TM OpTmizer TM T cell expansion SFM supplemented with approximately 3% CTS TM The final concentration of immune cell serum replacement (SR) (ThermoFisher Scientific) and 2-mercaptoethanol in the culture medium was 55 μM.

[0762] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., glutamine) 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 defined medium is supplemented with glutamine at a concentration of about 2 mM (i.e. ).

[0763] In some embodiments, the serum-free medium or defined 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 defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

[0764] In some embodiments, the defined medium described in International PCT Patent Publication No. WO / 1998 / 030679 (which is incorporated herein by reference) can be used for the present invention. In the publication, serum-free eukaryotic cell culture medium is described. Serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement that can support cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises the following or is obtained by combining one or more components selected from the following 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. In some embodiments, the defined medium also comprises L-glutamine, sodium bicarbonate and / or beta-mercaptoethanol. In some embodiments, the defined medium comprises albumin or albumin substitutes and one or more components selected from the following 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 defined 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 portion containing trace elements 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+In some embodiments, the basal cell culture medium is selected from the group consisting of: Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), basal medium of Eagle (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium and Iskoff's modified Dulbecco's medium.

[0765] In some embodiments, the concentration of glycine in the defined medium is in the range of 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, and the concentration of L-threonine is about 10 to 500 mg / L. g / L, L-tryptophan is at a concentration of about 2 to 110 mg / L, L-tyrosine is at a concentration of about 3 to 175 mg / L, L-valine is at a concentration of about 5 to 500 mg / L, thiamine is at a concentration of about 1 to 20 mg / L, reduced glutathione is at a concentration of about 1 to 20 mg / L, L-ascorbic acid-2-phosphate is at a concentration of about 1 to 200 mg / L, iron-saturated transferrin is at a concentration of about 1 to 50 mg / L, insulin is at a concentration of about 1 to 100 mg / L, sodium selenite is at a concentration of about 0.000001 to 0.0001 mg / L, and albumin (e.g. I) is at a concentration of about 5000 to 50,000 mg / L.

[0766] In some embodiments, the non-trace element portion components in the defined medium are present in the concentration ranges listed in the column entitled "Concentration Ranges in 1X Medium" in Table A below. In other embodiments, the non-trace element portion components in the defined medium are present in the final concentrations listed in the column entitled "Preferred Embodiments of 1X Medium" in Table A below. In other embodiments, the defined medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element portion components of the types and concentrations listed in the column entitled "Preferred Embodiments of Supplements" in Table A below.

[0767] Table A: Concentration of some components other than trace elements

[0768]

[0769] In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L or about 2.2 g / L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-mercaptoethanol (final concentration of about 100 μM).

[0770] In some embodiments, the defined 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) can be used in the present invention. In brief, RPMI or CTS TM OpTmizer TM Used as basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS TM Immune cell serum replacement.

[0771] In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container is unfiltered. Using unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In one embodiment, the cell culture medium in the first gas permeable container and / or the second gas permeable container lacks beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0772] In one embodiment, a rapid second expansion (including an expansion called REP) is performed and further comprises a step in which TILs with excellent tumor reactivity are selected. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with excellent tumor reactivity.

[0773] Optionally, cell viability assays can be performed using standard assays known in the art after rapid second amplification (including an amplification known as REP amplification). For example, a trypan blue exclusion assay can be performed on a sample of bulk TILs, which selectively marks dead cells and allows for viability assessment. In some embodiments, TIL samples can be calculated and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard cell counter, the K2 Image Cytometer automated cell counter protocol.

[0774] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of limited but large amounts of gene segments. These gene segments: V (variable region), D (diversity region), J (joining region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides a method for producing TILs that exhibit and increase the diversity of T cell repertoires. In some embodiments, the TILs obtained by the present method exhibit increased diversity of T cell repertoires. In some embodiments, the TILs obtained in the second amplification exhibit increased diversity of T cell repertoires. In some embodiments, increasing diversity is increasing immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, diversity is present in immunoglobulins, in immunoglobulin heavy chains. In some embodiments, diversity is present in immunoglobulins, in immunoglobulin light chains. In some embodiments, diversity is present in T cell receptors. In some embodiments, diversity is present 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 receptors (TCR) α and / or β increases. 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., TCR α / β) is increased.

[0775] In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or a second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or a second cell culture medium) comprises 6000 IU / mL IL-2, 30 ug / flask OKT-3, and 7.5×10 8In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or a second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs) as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or a second cell culture medium) comprises 6000 IU / mL IL-2, 30 ug / culture bottle OKT-3, and 5×10 8 Antigen presenting feeder cells (APCs).

[0776] In some embodiments, a rapid second amplification (e.g., according to FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) is carried out in a closed system bioreactor. In some embodiments, a closed system is used to expand TIL as described herein. In some embodiments, a bioreactor is used. In some embodiments, a 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.

[0777] 1. Feeder cells and antigen presenting cells

[0778] In one embodiment, the rapid second amplification procedure described herein (e.g., comprising a procedure such as that shown in FIG. 1 (particularly, e.g., Figure 1B and / or Figure 1C ) those described in step D and those referred to as REP) require excess feeder cells during REP TIL expansion and / or during rapid second expansion. In many embodiments, feeder cells are obtained from peripheral blood mononuclear cells (PBMCs) of standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

[0779] In general, allogeneic PBMCs are deactivated via irradiation or heat treatment and used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the replication inability of irradiated allogeneic PBMCs.

[0780] In some embodiments, if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells placed into culture on REP day 0 and / or second expansion day 0 (i.e., the start day of the second expansion), the PBMCs are considered replication incompetent and are acceptable for use in the TIL expansion procedures described herein.

[0781] In some embodiments, if the total number of viable cells at days 7 and 14 of culture in the presence of OKT3 and IL-2 is not increased from the initial number of viable cells placed in culture at REP day 0 and / or second expansion day 0 (i.e., the starting day of the second expansion), the PBMC is considered to be replication incompetent and acceptable for use in the TIL expansion procedure described herein. In some embodiments, PBMCs are cultured in the presence of 30ng / ml OKT3 antibodies and 3000IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 60ng / ml OKT3 antibodies and 6000IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 60ng / ml OKT3 antibodies and 3000IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30ng / ml OKT3 antibodies and 6000IU / ml IL-2.

[0782] In some embodiments, if the total number of viable cells at days 7 and 14 of culture in the presence of OKT3 and IL-2 is not increased from the initial number of viable cells placed in culture at REP day 0 and / or second expansion day 0 (i.e., the starting day of the second expansion), the PBMC is considered to be replication incompetent and acceptable for use in the TIL expansion program described herein. In some embodiments, PBMCs are cultured in the presence of 30 to 60 ng / ml OKT3 antibodies and 1000 to 6000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30 to 60 ng / ml OKT3 antibodies and 2000 to 5000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30 to 60 ng / ml OKT3 antibodies and 2000 to 4000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30 to 60 ng / ml OKT3 antibodies and 2500 to 3500 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30 to 60 ng / ml OKT3 antibody and 6000 IU / ml IL-2.

[0783] In some embodiments, antigen presenting feeder cells are PBMCs. In some embodiments, 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 amplification is about 1 to 10, about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400 or about 1 to 500. In one embodiment, the ratio of TIL to antigen presenting feeder cells in the second amplification is between 1 to 50 and 1 to 300. In one embodiment, the ratio of TIL to antigen presenting feeder cells in the second amplification is between 1 to 100 and 1 to 200.

[0784] In one embodiment, the second amplification procedure described herein requires about 5×10 8 Feeder cells and about 100×10 6 In one embodiment, the second expansion procedure described herein requires about 7.5×10 8 Feeder cells and about 100×10 6 In another embodiment, the second amplification procedure described herein requires about 5×10 8 Feeder cells and about 50×10 6 In another embodiment, the second expansion procedure described herein requires about 7.5×10 8 Feeder cells and about 50×10 6 In yet another embodiment, the second amplification procedure described herein requires about 5×10 8 Feeder cells and about 25×10 6 In yet another embodiment, the second expansion procedure described herein requires about 7.5×10 8 Feeder cells and about 25×10 6 In yet another embodiment, the rapid second expansion requires twice the number of feeder cells for the rapid second expansion. In yet another embodiment, when the priming first expansion described herein requires about 2.5×10 8 When the number of feeder cells is about 5×10 8 In yet another embodiment, when the priming first expansion described herein requires about 2.5×10 8 When the number of feeder cells is 10, the rapid second expansion requires about 7.5 × 10 8In yet another embodiment, the rapid second expansion requires twice (2.0X), 2.5X, 3.0X, 3.5X or 4.0X the number of feeder cells as the priming first expansion.

[0785] In one embodiment, the rapid second expansion procedure described herein requires excessive feeder cells during the rapid second expansion. In many embodiments, feeder cells are obtained from peripheral blood mononuclear cells (PBMC) of standard whole blood units of allogeneic healthy blood donors. PBMC is obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting (aAPC) cells are used instead of PBMC. In some embodiments, PBMC is added to the rapid second expansion at twice the concentration of PBMC added to the priming first expansion.

[0786] Generally, allogeneic PBMCs are deactivated via irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the Figures and Examples.

[0787] In one embodiment, artificial...

Claims

1. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising: (a) performing a priming first expansion to produce a second TIL population by culturing a first TIL population in a cell culture medium comprising IL-2, OKT-3 and antigen presenting cells (APCs), wherein the first TIL population can be obtained by processing a tumor sample from a tumor resected from a subject into a plurality of tumor fragments, wherein the priming first expansion is performed in a container comprising a first gas permeable surface area, wherein the priming first expansion is performed for a first period of 1 to 7 / 8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population; (b) performing a rapid second expansion by contacting the second TIL population with a cell culture medium of the second TIL population containing additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs in the rapid second expansion is at least twice the number of APCs in step (a), wherein the rapid second expansion is performed for a second period of 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas permeable surface area; and (c) harvesting the therapeutic TIL population obtained from step (b). 2 . The method according to claim 1 , wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is 1.5:1 to 100:

1.

3. The method according to claim 1, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is 50:

1.

4. The method according to claim 1, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is 25:

1. 5 . The method of claim 1 , wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is 20:

1. 6 . The method of claim 1 , wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is 10:

1.

7. The method of claim 1, wherein the second TIL population is at least 50 times greater in number than the first TIL population.

8. The method of claim 1, wherein the method comprises, after the step of harvesting the therapeutic TIL population, performing the following additional step: transferring the harvested therapeutic TIL population to an infusion bag.

9. The method of claim 1, wherein the plurality of tumor fragments are distributed in a plurality of separate containers, in each of the separate containers, the second TIL population is obtained from the first TIL population in the priming first expansion step, and the third TIL population is obtained from the second TIL population in the rapid second expansion step, and wherein the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to produce a harvested TIL population.

10. The method of claim 9, wherein the plurality of separate containers comprises at least two separate containers.

11. The method of claim 9, wherein the plurality of separate containers comprises two to twenty separate containers.

12. The method of claim 9, wherein the plurality of separate containers comprises two to ten separate containers.

13. The method of claim 9, wherein the plurality of separate containers comprises two to five separate containers.

14. The method of claim 9, wherein each of the separate containers comprises a first gas permeable surface area.

15. The method of any one of claims 1 to 14, wherein the plurality of tumor fragments are distributed in a single container.

16. The method of claim 15, wherein the single container comprises a first gas permeable surface region.

17. The method according to any one of claims 1 to 14, wherein in the priming first expansion step, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered on the first gas permeable surface area with an average thickness of 1 cell layer to 3 cell layers.

18. The method of claim 17, wherein the APCs are layered on the first gas permeable surface area at an average thickness of 1.5 cell layers to 2.5 cell layers.

19. The method of claim 17, wherein the APCs are layered on the first gas permeable surface area with an average thickness of 2 cell layers.

20. The method of claim 17, wherein in the rapid second expansion step, the APCs are layered on the first gas permeable surface area in a thickness of 3 to 5 cell layers.

21. A method according to any one of claims 1 to 14, wherein in the priming first amplification step, the priming first amplification is performed in a first container comprising a first gas permeable surface area, and in the rapid second amplification step, the rapid second amplification is performed in a second container comprising a second gas permeable surface area.

22. The method of claim 21, wherein the second container is larger than the first container.

23. The method of claim 21, wherein in the rapid second expansion step, the APCs are layered on the second gas permeable surface area with an average thickness of 3 to 5 cell layers.

24. The method according to any one of claims 1 to 14, wherein for each container in which the priming first expansion is performed on a first TIL population, the rapid second expansion is performed on the second TIL population generated from such first TIL population in the same container.

25. The method of claim 24, wherein each container comprises a first gas permeable surface region.

26. The method of any one of claims 1 to 14, wherein for each container in which the priming first expansion is performed on a first TIL population, in the priming first expansion step, the first container comprises a first surface area, the cell culture medium comprises antigen presenting cells (APCs), and the APCs are layered on the first gas permeable surface area, and wherein the ratio of the average number of APC layers layered in the priming first expansion step to the average number of APC layers layered in the rapid second expansion step is in the range of 1:1.1 to 1:

10.

27. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.2 to 1:

8.

28. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.3 to 1:

7.

29. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.4 to 1:

6.

30. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.5 to 1:

5.

31. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.6 to 1:

4.

32. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.7 to 1:3.

5.

33. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.8 to 1:

3.

34. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is in the range of 1:1.9 to 1:2.

5.

35. The method of claim 26, wherein the ratio of the average number of APCs stacked in the priming first amplification step to the average number of APCs stacked in the rapid second amplification step is 1:

2.

36. The method of any one of claims 1 to 14, wherein after 2 to 3 days in the rapid second expansion step, the cell culture medium is supplemented with additional IL-2.

37. The method of claim 36, wherein the method further comprises cryopreserving the harvested TIL population in the step of harvesting the therapeutic TIL population using a cryopreservation process.

38. The method according to any one of claims 9 to 14, further comprising the step of freezing the infusion bag.

39. The method of claim 38, wherein the cryopreservation process is performed using a 1:1 ratio of harvested TIL population to cryopreservation medium.

40. The method of any one of claims 1 to 14, wherein the antigen presenting cells are peripheral blood mononuclear cells (PBMC).

41. The method of claim 40, wherein the PBMCs are irradiated and allogeneic.

42. The method of claim 40, wherein in the priming first expansion step, the cell culture medium comprises peripheral blood mononuclear cells (PBMCs), and wherein the total number of PBMCs added to the cell culture medium in the priming first expansion step is 2.5×10 8 and / or wherein in the rapid second expansion step, the antigen presenting cells (APCs) in the cell culture medium are peripheral blood mononuclear cells (PBMCs), and wherein the total number of PBMCs added to the cell culture medium in the rapid second expansion step is 5×10 8 indivual.

43. The method of any one of claims 1 to 14, wherein the antigen presenting cell is an artificial antigen presenting cell.

44. The method of any one of claims 1 to 14, wherein the harvesting in the step of harvesting the therapeutic TIL population is performed using a membrane-based cell processing system.

45. The method of claim 44, wherein the harvesting is performed using the LOVO cell processing system.

46. ​​The method of any one of claims 1 to 14, wherein the plurality of segments comprises 30 to 60 segments having a total volume of 1300 mm 3 Up to 1500 mm 3 .

47. The method of claim 46, wherein in the priming first amplification step the plurality of fragments comprises 60 fragments per container, wherein each fragment has 27 mm 3 volume.

48. The method of claim 46, wherein the total mass of the plurality of fragments is 1 gram to 1.5 grams.

49. The method of any one of claims 1 to 14, wherein the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri Cell Bag.

50. The method of any one of claims 1 to 14, wherein the IL-2 concentration is 10,000 IU / mL to 5,000 IU / mL.

51. The method of any one of claims 9 to 14, wherein in the step of transferring the harvested therapeutic TIL population to an infusion bag, the infusion bag is an infusion bag containing HypoThermosol.

52. The method of any one of claims 39 to 42, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO).

53. The method of claim 52, wherein the cryopreservation medium comprises 7% to 10% DMSO.

54. The method of any one of claims 1 to 14, wherein the first time period of the priming first amplification step and the second time period of the rapid second amplification step are each individually performed within a time period of 5 days, 6 days or 7 days.

55. The method of claim 54, wherein the second period of time of the rapid second amplification step is performed over a period of 7 days, 8 days or 9 days.

56. The method of any one of claims 1 to 14, wherein the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed within a time period of 14 to 16 days.

57. The method of any one of claims 1 to 14, wherein the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed within a period of 16 days.

58. The method of any one of claims 1 to 14, wherein the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed within a period of 15 days.

59. The method of any one of claims 1 to 14, wherein the steps of said priming first expansion to said harvesting of said therapeutic TIL population are performed within a period of 14 days.

60. The method of any one of claims 1 to 14, further comprising the step of cryopreserving the harvested therapeutic TIL population using a cryopreservation process, wherein the steps of priming first expansion to harvesting the therapeutic TIL population and cryopreservation are performed within 16 days or less.

61. The method of any one of claims 1 to 14, wherein the therapeutic TIL population harvested in the step of harvesting the therapeutic TIL population comprises sufficient TILs for a therapeutically effective dose of the TILs.

62. The method of claim 61, wherein a sufficient number of TILs for a therapeutically effective dose is 2.3×10 10 to 13.7×10 10 indivual.

63. The method of any one of claims 1 to 14, wherein the third TIL population in the rapid second expansion step provides increased efficacy, increased interferon-γ production and / or increased polyclonality.

64. The method of claim 63, wherein the third TIL population in the rapid second expansion step provides at least 1 to 5 or more times the production of interferon-γ compared to TIL prepared by a process longer than 18 days.

65. The method of claim 63, wherein the effector T cells and / or central memory T cells obtained from the third TIL population in the rapid second expansion step exhibit increased expression of CD8 and CD28 relative to the effector T cells and / or central memory T cells obtained from the second TIL population in the priming first expansion step.

66. Expanded tumor infiltrating lymphocytes (TIL) for use in a method of treating a subject having cancer, the method comprising administering the expanded tumor infiltrating lymphocytes (TIL), the method comprising: (a) obtaining and / or receiving a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) performing a priming first expansion to produce a second TIL population by culturing the first TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs), wherein the priming first expansion is performed in a container comprising a first gas permeable surface area, wherein the priming first expansion is performed for 1 to 7 / 8 days to obtain the second TIL population, wherein the second TIL population is at least 50 times greater in number than the first TIL population; (c) performing a rapid second 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 rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second 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; and (f) administering a therapeutically effective dose of the TILs from step (e) to the subject.

67. The expanded tumor infiltrating lymphocytes (TILs) of claim 66, wherein a sufficient number of TILs for administering a therapeutically effective dose in step (f) is 2.3×10 10 to 13.7×10 10 indivual.

68. The expanded tumor infiltrating lymphocytes (TILs) of claim 66, wherein the antigen presenting cells (APCs) are PBMCs.

69. The expanded tumor infiltrating lymphocytes (TIL) of claim 68, wherein the patient has been administered a non-myeloablative lymphocyte depletion regimen prior to administering the therapeutically effective dose of TIL cells in step (f).

70. The expanded tumor infiltrating lymphocytes (TILs) of claim 69, wherein the non-myeloablative lymphocyte depletion regimen comprises administering a dose of 60 mg / m 2 Cyclophosphamide was administered daily for two days followed by 25 mg / m 2 / day of fludarabine for five days in steps.

71. According to the expanded tumor infiltrating lymphocytes (TIL) described in any one of claims 66 to 70, the method also includes a step of treating the patient with a high-dose IL-2 regimen on the day after the TIL cells are administered to the patient in step (f).

72. The expanded tumor infiltrating lymphocytes (TILs) of claim 71, wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15-minute intravenous push every eight hours until tolerated.

73. The expanded tumor infiltrating lymphocytes (TILs) of any one of claims 66 to 70, wherein the third TIL population in step (c) provides increased efficacy, increased interferon-γ production and / or increased polyclonality.

74. The expanded tumor infiltrating lymphocytes (TILs) of claim 73, wherein the third TIL population in step (c) provides at least 1 to 5 times or more interferon-γ production compared to TILs prepared by a process longer than 16 days.

75. The expanded tumor infiltrating lymphocytes (TILs) of claim 73, wherein the effector T cells and / or central memory T cells obtained from the third TIL population in step (c) exhibit increased expression of CD8 and CD28 relative to the effector T cells and / or central memory T cells obtained from the second cell population in step (b).

76. The expanded tumor infiltrating lymphocytes (TILs) of any one of claims 66 to 70, wherein the cancer is a solid tumor.

77. The expanded tumor infiltrating lymphocytes (TILs) of any one of claims 66 to 70, 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 papilloma virus, head and neck cancer, glioblastoma, gastrointestinal cancer, kidney cancer.

78. The expanded tumor infiltrating lymphocytes (TILs) of claim 77, wherein the head and neck cancer comprises head and neck squamous cell carcinoma (HNSCC), the glioblastoma comprises GBM, and the renal cancer comprises renal cell carcinoma.

79. The expanded tumor infiltrating lymphocytes (TILs) of any one of claims 66 to 70, wherein the container is a closed container.

80. The expanded tumor infiltrating lymphocytes (TILs) of claim 79, wherein the sealed container comprises GREX-100 or GREX-500; G container or GREX-10.

81. A therapeutic tumor infiltrating lymphocyte (TIL) population obtained by the method of any one of claims 1 to 65, wherein the therapeutic TIL population: a) the TILs are capable of producing at least 1-fold more interferon-γ than TILs prepared by a process wherein the first expansion of the TILs is performed in the absence of any added antigen presenting cells (APCs); b) the TILs are capable of producing at least 1-fold more interferon-γ compared to TILs prepared by a process wherein the first expansion of the TILs is performed without any added OKT3; or c) TILs were able to produce at least 1-fold more interferon-γ compared to TILs prepared by a process in which the first expansion of TILs was performed without added antigen presenting cells (APCs) and without added OKT3.

82. A tumor infiltrating lymphocyte (TIL) composition comprising the therapeutic TIL population according to claim 32 and a pharmaceutically acceptable carrier.

83. The tumor infiltrating lymphocyte (TIL) composition of claim 82, wherein the composition comprises a therapeutic TIL population according to claim 32 and a cryopreservation medium.

84. The tumor infiltrating lymphocyte (TIL) composition of claim 83, wherein the cryopreservation medium contains 7% to 10% DMSO.

85. A tumor infiltrating lymphocyte (TIL) composition according to claim 84, for use in a method of treating cancer in a subject, the method comprising administering a therapeutically effective dose of the TIL composition to the subject.

86. The tumor infiltrating lymphocyte (TIL) composition of claim 85, wherein the cancer is a solid tumor.

87. The tumor infiltrating lymphocyte (TIL) composition of claim 85, 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 papilloma virus, head and neck cancer, glioblastoma, gastrointestinal cancer, kidney cancer, hypermutation cancer, and pediatric hypermutation cancer.

88. The tumor infiltrating lymphocyte (TIL) composition of claim 87, wherein the head and neck cancer comprises head and neck squamous cell carcinoma (HNSCC), the glioblastoma comprises GBM, and the renal cancer comprises renal cell carcinoma.

89. The method of any one of claims 1 to 14, wherein the tumor sample is one or more small biopsies, core biopsies, or needle biopsies of the tumor of the subject.

90. The expanded tumor infiltrating lymphocytes (TILs) of any one of claims 66 to 70, wherein the therapeutic TIL population exhibits: (a) CDR diversity is increased compared to TILs prepared by a method without adding any antigen presenting cells (APCs) during the first expansion of TILs; (b) the CDR diversity is increased compared to TILs prepared by a method without adding any OKT3 during the first expansion of TILs; or (c) CDR diversity increased compared to TIL prepared by a method without adding antigen presenting cells (APCs) and OKT3 during the first expansion of TIL.

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