Method for expanding tumor-infiltrating lymphocytes using an engineered cytokine receptor pair and its use

Abstract

The present invention provides improved and / or shortened methods for expanding TILs and generating therapeutic TIL populations, including novel methods for expanding TIL populations in a closed system that result in improved efficacy, improved phenotype, and increased metabolic health of TILs in a shorter period of time, while allowing for reduced microbial contamination and reduced costs. Methods for expanding TILs that express orthogonal cytokine receptors are provided. Such TILs find use in therapeutic treatment regimens.

Description

Technical Field

[0001] Cross - reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 782,330, filed on December 19, 2018, the entire disclosure of which is hereby incorporated by reference.

Background Art

[0002] Background of the Invention

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

[0003]

[0003] The current TIL treatment protocol utilizes the administration of IL-2 (aldesleukin) after injecting TILs into patients. The safety profile of aldesleukin can negatively impact the overall safety of TIL therapy. However, without being bound by any theory, the use of modified TILs containing engineered IL-2 receptors and mutant IL-2 proteins can avoid the overall immune system effects of aldesleukin and potentially improve the treatment outcome of TIL therapy by binding to the receptor.

Summary of the Invention

Means for Solving the Problems

[0004] Summary of the Invention

[0004] The present invention provides an improved and / or shortened method for expanding TILs and generating a therapeutic TIL population.

[0005]

[0005] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) performing a first priming expansion culture to generate a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs), wherein the first priming expansion culture is performed in a container comprising a first gas-permeable surface area, the first priming expansion culture is performed over a first period of about 1 to 7 days to obtain the second TIL population, and the second TIL population is greater in number than the first TIL population; (c) By supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APC, performing a second rapid expansion culture to generate a third TIL population, wherein the number of APC added in the second rapid expansion culture is at least twice the number of APC added in step (b), the second rapid expansion culture is performed over a second period of about 1 to 11 days to obtain the third TIL population, the third TIL population is a therapeutic TIL population, and the second rapid expansion culture is performed in a container containing a second gas permeable surface area; (d) Manipulating the TIL generated in step (c) to express an orthogonal IL-2Rβ; (e) Recovering the therapeutic TIL population obtained from step (d); and (f) Transferring the recovered TIL population from step (e) to an infusion bag comprising.

[0006]

[0006] In some embodiments, the present invention provides a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, the method comprising (a) Obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) Performing a first primary stimulation expansion culture to generate a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and optionally antigen presenting cells (APC), wherein the first primary stimulation expansion culture is performed over a first period of about 1 to 7 days to obtain the second TIL population, and the second TIL population is greater in number than the first TIL population; (c) Performing a second rapid expansion culture to generate a third TIL population by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3, and APC, wherein the second rapid expansion culture is performed over a second period of about 1 to 11 days to obtain the third TIL population, and the third TIL population is a therapeutic TIL population; (d) Manipulating the TILs generated in step (c) so as to express orthogonal IL-2Rβ; and (e) Recovering the therapeutic TIL population obtained from step (d) comprises.

[0007]

[0007] In some embodiments, the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium of step (c) is greater than the number of APCs in the culture medium of step (b).

[0008]

[0008] In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) Culturing a first TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs), the first TIL population being obtainable by treating a tumor sample from a tumor excised from a subject into a plurality of tumor fragments, thereby performing a first primary stimulation expansion culture to generate a second TIL population, wherein the first primary stimulation expansion culture is performed in a container comprising a first gas-permeable surface area, the first primary stimulation expansion culture is performed over a first period of about 1 to 7 days to obtain the second TIL population, and the second TIL population is greater in number than the first TIL population; (b) Performing a second rapid expansion culture to generate a third TIL population by contacting the second TIL population with a cell culture medium of the second TIL population having additional IL-2, OKT-3, and APCs, wherein the number of APCs in the second rapid expansion culture is at least twice the number of APCs in step (a), the second rapid expansion culture is performed over a second period of about 1 to 11 days to obtain the third TIL population, the third TIL population is the therapeutic TIL population, and the second rapid expansion culture is performed in a container comprising a second gas-permeable surface area; (c) Manipulating the TILs generated in step (b) so as to express orthogonal IL-2Rβ; and (d) Recovering the therapeutic TIL population obtained from step (c) comprises.

[0009]

[0009] In some embodiments, the present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) performing a first priming expansion culture by culturing a first TIL population in a cell culture medium comprising IL-2, OKT-3, and optionally antigen-presenting cells (APCs), to generate a second TIL population, wherein the first priming expansion culture is performed over a first period of about 1 to 7 days to obtain the second TIL population, and the second TIL population is greater in number than the first TIL population; (b) engineering the TILs generated in step (a) to express an orthogonal IL-2Rβ; (c) performing a second rapid expansion culture by contacting the second TIL population with a cell culture medium comprising orthogonal IL-2, OKT-3, and APCs, to generate a third TIL population, wherein the second rapid expansion culture is performed over a second period of about 1 to 11 days to obtain the third TIL population, and the third TIL population is the therapeutic TIL population; and (d) recovering the therapeutic TIL population obtained from step (c). comprises.

[0010]

[0010] In some embodiments, in step (b), the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium of step (c) is greater than the number of APCs in the culture medium of step (b).

[0011]

[0011] In some embodiments, the ratio of the number of APCs in the second rapid expansion culture to the number of APCs in the first priming expansion culture is in the range of about 1.5:1 to about 20:1.

[0012]

[0012] In some embodiments, the ratio is in the range of about 1.5:1 to about 10:1.

[0013]

[0013] In some embodiments, the ratio is in the range of about 2:1 to about 5:1.

[0014]

[0014] In some embodiments, the ratio is in the range of about 2:1 to about 3:1.

[0015]

[0015] In some embodiments, the ratio is 2:1.

[0016]

[0016] In some embodiments, the number of APCs in the first priming expansion culture is about 1.0×10 6 APCs / cm 2 to about 4.5×10 6 APCs / cm 2 and the number of APCs in the second rapid expansion culture is about 2.5×10 6 APCs / cm 2 to about 7.5×10 6 APCs / cm 2 respectively.

[0017]

[0017] In some embodiments, the number of APCs in the first priming expansion culture is about 1.5×10 6 APCs / cm 2 to about 3.5×10 6 APCs / cm 2 and the number of APCs in the second rapid expansion culture is about 3.5×10 6 APCs / cm 2 to about 6.0×10 6 APCs / cm 2 respectively.

[0018]

[0018] In some embodiments, the number of APCs in the first priming expansion culture is about 2.0×10 6 APCs / cm 2 to about 3.0×10 6 APCs / cm 2 and the number of APCs in the second rapid expansion culture is about 4.0×10 6 APCs / cm 2 to about 5.5×106 APC / cm 2 is in the range of.

[0019]

[0019] In some embodiments, the number of APCs in the first priming expansion culture is about 1×10 8 APCs to about 3.5×10 8 APCs, and the number of APCs in the second rapid expansion culture is about 3.5×10 8 APCs to about 1×10 9 APCs.

[0020]

[0020] In some embodiments, the number of APCs in the first priming expansion culture is about 1.5×10 8 APCs to about 3×10 8 APCs, and the number of APCs in the second rapid expansion culture is about 4×10 8 APCs to about 7.5×10 8 APCs.

[0021]

[0021] In some embodiments, the number of APCs in the first priming expansion culture is about 2×10 8 APCs to about 2.5×10 8 APCs, and the number of APCs in the second rapid expansion culture is about 4.5×10 8 APCs to about 5.5×10 8 APCs.

[0022]

[0022] In some embodiments, about 2.5×10 8 APCs are added to the first priming expansion culture, and 5×10 8 APCs are added to the second rapid expansion culture.

[0023]

[0023] 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.

[0024]

[0024] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 50:1.

[0025]

[0025] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 25:1.

[0026]

[0026] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 20:1.

[0027]

[0027] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 10:1.

[0028]

[0028] In some embodiments, the second TIL population is at least 50 times greater in number than the first TIL population.

[0029]

[0029] In some embodiments, the method includes, after the step of collecting the therapeutic TIL population, performing an additional step of transferring the collected therapeutic TIL population to an infusion bag.

[0030]

[0030] In some embodiments, the plurality of tumor fragments are distributed into a plurality of separate containers, and in each of the separate containers, the second TIL population is obtained from the first TIL population in the first primary stimulation expansion culture step, and the third TIL population is obtained from the second TIL population in the second rapid expansion culture step, and the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to yield the collected TIL population.

[0031]

[0031] In some embodiments, the plurality of separate containers includes at least two separate containers.

[0032] In some embodiments, the plurality of separate containers includes at least 2 to 20 separate containers.

[0033] In some embodiments, the plurality of separate containers includes at least 2 to 10 separate containers.

[0034] In some embodiments, the plurality of separate containers includes at least 2 to 5 separate containers.

[0035] In some embodiments, each of the separate containers includes a first gas-permeable surface area.

[0036] In some embodiments, the plurality of tumor fragments are distributed within a single container.

[0037] In some embodiments, the single container includes a first gas-permeable surface area.

[0038] In some embodiments, in the step of the first priming expansion culture, the cell culture medium includes antigen-presenting cells (APCs), and the APCs are stacked on the first gas-permeable surface area with an average thickness of about 1 cell layer to about 3 cell layers.

[0039] In some embodiments, in the step of the first priming expansion culture, the APCs are stacked on the first gas-permeable surface area with an average thickness of about 1.5 cell layers to about 2.5 cell layers.

[0040] In some embodiments, in the step of the first priming expansion culture, the APCs are stacked on the first gas-permeable surface area with an average thickness of about 2 cell layers.

[0041] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the first gas-permeable surface area with a thickness of about 3 cell layers to about 5 cell layers.

[0042]

[0042] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the first gas permeable surface area with a thickness of about 3.5 cell layers to about 4.5 cell layers.

[0043]

[0043] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the first gas permeable surface area with a thickness of about 4 cell layers.

[0044]

[0044] In some embodiments, in the step of the first initial stimulation expansion culture, the first initial stimulation expansion culture is carried out in a first container containing the first gas permeable surface area, and in the step of the second rapid expansion culture, the second rapid expansion culture is carried out in a second container containing the second gas permeable surface area.

[0045]

[0045] In some embodiments, the second container is larger than the first container.

[0046]

[0046] In some embodiments, in the step of the first initial stimulation expansion culture, the cell culture medium contains antigen-presenting cells (APCs), and the APCs are stacked on the first gas permeable surface area with an average thickness of about 1 cell layer to about 3 cell layers.

[0047]

[0047] In some embodiments, in the step of the first initial stimulation expansion culture, the APCs are stacked on the first gas permeable surface area with an average thickness of about 1.5 cell layers to about 2.5 cell layers.

[0048]

[0048] In some embodiments, in the step of the first initial stimulation expansion culture, the APCs are stacked on the first gas permeable surface area with an average thickness of about 2 cell layers.

[0049]

[0049] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the second gas permeable surface area with an average thickness of about 3 cell layers to about 5 cell layers.

[0050]

[0050] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the second gas-permeable surface area with an average thickness of about 3.5 cell layers to about 4.5 cell layers.

[0051]

[0051] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the second gas-permeable surface area with an average thickness of about 4 cell layers.

[0052]

[0052] In some embodiments, for each container in which the first primary stimulation expansion culture is performed on the first TIL population, the second rapid expansion culture is performed in the same container on the second TIL population generated from such first TIL population.

[0053]

[0053] In some embodiments, each container includes a first gas-permeable surface area.

[0054]

[0054] In some embodiments, in the step of the first primary stimulation expansion culture, the cell culture medium includes antigen-presenting cells (APCs), and the APCs are stacked on the first gas-permeable surface area with an average thickness of about 1 cell layer to about 3 cell layers.

[0055]

[0055] In some embodiments, in the step of the first primary stimulation expansion culture, the APCs are stacked on the first gas-permeable surface area with an average thickness of about 1.5 cell layers to about 2.5 cell layers.

[0056]

[0056] In some embodiments, in the step of the first primary stimulation expansion culture, the APCs are stacked on the first gas-permeable surface area with an average thickness of about 2 cell layers.

[0057]

[0057] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the first gas-permeable surface area with an average thickness of about 3 cell layers to about 5 cell layers.

[0058]

[0058] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the first gas-permeable surface area with an average thickness of about 3.5 cell layers to about 4.5 cell layers.

[0059]

[0059] In some embodiments, in the step of the second rapid expansion culture, the APCs are stacked on the first gas-permeable surface area with an average thickness of about 4 cell layers.

[0060]

[0060] In some embodiments, for each container in which the first primary stimulation expansion culture is performed on the first TIL population in the step of the first primary stimulation expansion culture, the first container includes a first surface area, the cell culture medium includes antigen-presenting cells (APCs), and the APCs are stacked on the first gas-permeable surface area, and the ratio of the average number of layers of APCs stacked in the step of the first primary stimulation expansion culture to the average number of layers of APCs stacked in the step of the second rapid expansion culture is in the range of about 1:1.1 to about 1:10.

[0061]

[0061] In some embodiments, the ratio of the average number of layers of APCs stacked in the step of the first primary stimulation expansion culture to the average number of layers of APCs stacked in the step of the second rapid expansion culture is in the range of about 1:1.2 to about 1:8.

[0062]

[0062] In some embodiments, the ratio of the average number of layers of APCs stacked in the step of the first primary stimulation expansion culture to the average number of layers of APCs stacked in the step of the second rapid expansion culture is in the range of about 1:1.3 to about 1:7.

[0063]

[0063] In some embodiments, the ratio of the average number of layers of APCs stacked in the step of the first primary stimulation expansion culture to the average number of layers of APCs stacked in the step of the second rapid expansion culture is in the range of about 1:1.4 to about 1:6.

[0064] In some embodiments, the ratio of the average number of layers of APCs stacked in the first priming expansion culture step to the average number of layers of APCs stacked in the second rapid expansion culture step is in the range of about 1:1.5 to about 1:5.

[0065]

[0065] In some embodiments, the ratio of the average number of layers of APCs stacked in the first priming expansion culture step to the average number of layers of APCs stacked in the second rapid expansion culture step is in the range of about 1:1.6 to about 1:4.

[0066]

[0066] In some embodiments, the ratio of the average number of layers of APCs stacked in the first priming expansion culture step to the average number of layers of APCs stacked in the second rapid expansion culture step is in the range of about 1:1.7 to about 1:3.5.

[0067]

[0067] In some embodiments, the ratio of the average number of layers of APCs stacked in the first priming expansion culture step to the average number of layers of APCs stacked in the second rapid expansion culture step is in the range of about 1:1.8 to about 1:3.

[0068]

[0068] In some embodiments, the ratio of the average number of layers of APCs stacked in the first priming expansion culture step to the average number of layers of APCs stacked in the second rapid expansion culture step is in the range of about 1:1.9 to about 1:2.5.

[0069]

[0069] In some embodiments, the ratio of the average number of layers of APCs stacked in the first priming expansion culture step to the average number of layers of APCs stacked in the second rapid expansion culture step is about 1:2.

[0070]

[0070] In some embodiments, 2 to 3 days after the second rapid expansion culture step, the cell culture medium is supplemented with additional IL-2.

[0071]

[0071] In some embodiments, it further includes cryopreserving the collected TIL population in the step of collecting the therapeutic TIL population using a cryopreservation process.

[0072]

[0072] In some embodiments, it further includes the step of cryopreserving the infusion bag.

[0073]

[0073] In some embodiments, the cryopreservation process herein is carried out using a 1:1 ratio of the collected TIL population to the cryopreservation medium.

[0074]

[0074] In some embodiments, the antigen-presenting cell is a peripheral blood mononuclear cell (PBMC).

[0075]

[0075] In some embodiments, the PBMC is irradiated and allogeneic.

[0076]

[0076] In some embodiments, in the step of the first priming expansion culture, the cell culture medium contains peripheral blood mononuclear cells (PBMC), and the total number of PBMC in the cell culture medium in the step of the first priming expansion culture is 2.5×10 8 cells.

[0077]

[0077] In some embodiments, in the step of the second rapid expansion culture, the antigen-presenting cell (APC) in the cell culture medium is a peripheral blood mononuclear cell (PBMC), and the total number of PBMC added to the cell culture medium in the step of the second rapid expansion culture is 5×10 8 cells.

[0078]

[0078] In some embodiments, the antigen-presenting cell is an artificial antigen-presenting cell.

[0079]

[0079] In some embodiments, the collection in the step of collecting the therapeutic TIL population is carried out using a membrane-based cell processing system.

[0080] In some embodiments, the collection in step (d) is performed using a LOVO cell processing system.

[0081]

[0081] In some embodiments, the plurality of fragments includes about 60 fragments per container in the step of the first priming expansion culture, and each fragment has a volume of about 27 mm 3 and has a volume of.

[0082]

[0082] In some embodiments, the plurality of fragments includes about 30 to about 60 fragments having a total volume of about 1300 mm 3 to about 1500 mm 3 and having a total volume of.

[0083]

[0083] In some embodiments, the plurality of fragments includes about 50 fragments having a total volume of about 1350 mm 3 and having a total volume of.

[0084]

[0084] In some embodiments, the plurality of fragments includes about 50 fragments having a total mass of about 1 gram to about 1.5 grams.

[0085]

[0085] In some embodiments, the cell culture medium is provided in a container selected from the group consisting of a G container and an Xuri cell culture bag.

[0086]

[0086] In some embodiments, the IL-2 concentration is about 10,000 IU / mL to about 5,000 IU / mL.

[0087]

[0087] In some embodiments, the IL-2 concentration is about 6,000 IU / mL.

[0088]

[0088] In some embodiments, the infusion bag in the step of transferring the recovered therapeutic TIL population to an infusion bag is an infusion bag containing HypoThermosol.

[0089]

[0089] In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO).

[0090]

[0090] In some embodiments, the cryopreservation medium contains 7% - 10% DMSO.

[0091]

[0091] In some embodiments, the first period in the step of the first initial stimulation expansion culture and the second period in the step of the second rapid expansion culture are each individually carried out within a period of 5 days, 6 days or 7 days.

[0092]

[0092] In some embodiments, the first period in the step of the first initial stimulation expansion culture is carried out within a period of 5 days, 6 days or 7 days.

[0093]

[0093] In some embodiments, the second period in the step of the second rapid expansion culture is carried out within a period of 7 days, 8 days or 9 days.

[0094]

[0094] In some embodiments, the first period in the step of the first initial stimulation expansion culture and the second period in the step of the second rapid expansion culture are each individually carried out within a period of 7 days.

[0095]

[0095] In some embodiments, the steps from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population are carried out within a period of about 14 days to about 16 days.

[0096]

[0096] In some embodiments, the steps from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population are carried out within a period of about 15 days to about 16 days.

[0097]

[0097] In some embodiments, the steps from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population are carried out within a period of about 14 days.

[0098]

[0098] In some embodiments, the steps from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population are carried out within a period of about 15 days.

[0099]

[0099] In some embodiments, the steps from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population are carried out within a period of about 16 days.

[0100]

[0100] In some embodiments, the method further includes the step of cryopreserving the recovered therapeutic TIL population using a cryopreservation process, and the steps from the first initial stimulation expansion culture to the recovery and cryopreservation of the therapeutic TIL population are carried out within 16 days or less.

[0101]

[0101] In some embodiments, the therapeutic TIL population recovered in the step of recovering the therapeutic TIL population contains sufficient TILs for a therapeutically effective dose of TIL.

[0102]

[0102] In some embodiments, the number of sufficient TILs for a therapeutically effective dose is about 2.3×10 10 ~about 13.7×10 10 cells.

[0103]

[0103] In some embodiments, the third TIL population in the step of the second rapid expansion culture provides increased efficacy, increased interferon gamma production and / or increased polyclonality.

[0104]

[0104] In some embodiments, the third TIL population in the step of the second rapid expansion culture provides at least 1 to 5 times or more interferon gamma production compared to TILs prepared by a process longer than 16 days.

[0105] In some embodiments, the effector T cells and / or central memory T cells obtained from the third TIL population in the step of the second rapid expansion culture exhibit increased CD8 and CD28 expression compared to the effector T cells and / or central memory T cells obtained from the second TIL population in the step of the first initial stimulation expansion culture.

[0106] In some embodiments, the therapeutic TIL population from the step of harvesting the therapeutic TIL population is infused into the patient.

[0107] In some embodiments, the present invention provides a method of treating a subject having cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TIL), and administering expanded tumor-infiltrating lymphocytes (TIL) (a) obtaining a first TIL population from a tumor excised from the subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) performing a first initial stimulation expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APC) to generate a second TIL population, the first initial stimulation expansion culture being performed in a container containing a first gas-permeable surface area, the first initial stimulation expansion culture being performed over about 1 to 7 days to obtain the second TIL population, the second TIL population being at least 50 times more numerous than the first TIL population; (c) performing a second rapid expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and APC to generate a third TIL population, the number of APC added to the second rapid expansion culture being at least twice the number of APC added in step (b), the second rapid expansion culture being performed over about 1 to 11 days to obtain the third TIL population, the third TIL population being the therapeutic TIL population, the second rapid expansion culture being performed in a container containing a second gas-permeable surface area; (d) Recovering the therapeutic TIL population obtained from step (c); (e) Manipulating the TILs to express orthogonal IL-2Rβ; (f) Transferring the recovered TIL population from step (d) to an infusion bag; and (g) Administering a therapeutically effective dose of the TILs from step (f) to the subject comprising.

[0108]

[0108] In some embodiments, the number of TILs sufficient to administer the therapeutically effective dose in step (g) is about 2.3×10 10 ~ about 13.7×10 10 cells.

[0109]

[0109] In some embodiments, the antigen-presenting cell (APC) is PBMC.

[0110]

[0110] In some embodiments, a myeloablative lymphodepletion regimen is administered to the patient before administering the therapeutically effective dose of TIL cells in step (g).

[0111]

[0111] In some embodiments, the myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m 2 / day for 2 days, followed by administering fludarabine at a dose of 25 mg / m 2 / day for 5 days.

[0112]

[0112] In some embodiments, the method further comprises treating the patient with a high-dose IL-2 regimen starting on the day after administering the TIL cells to the patient in step (g).

[0113]

[0113] In some embodiments, the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15-minute bolus intravenous infusion every 8 hours up to the tolerated volume.

[0114]

[0114] In some embodiments, the method further includes treating the patient with a high-dose IL-2 regimen beginning the day after administering the TIL cells to the patient in step (g), wherein the IL-2 is orthogonal IL-2.

[0115]

[0115] In some embodiments, the high dose IL-2 regimen comprises 600,000 or 720,000 IU / kg of orthogonal IL-2 administered as a 15 minute bolus intravenous infusion every 8 hours to tolerated dose.

[0116] In some embodiments, high dose orthogonal IL-2 is administered starting the day after administration of the therapeutic population in step (g). In some embodiments, the high dose orthogonal IL-2 regimen comprises 600,000 or 720,000 IU / kg administered as a 15 minute bolus intravenous infusion every 8 hours to a tolerated dose.

[0117] In some embodiments, the third population of TILs in step (b) provides increased efficacy, increased interferon gamma production, and / or increased polyclonality.

[0118]

[0118] In some embodiments, the third population of TILs in step (c) provides at least 1-fold to 5-fold more interferon gamma production compared to TILs prepared by a process longer than 16 days.

[0119]

[0119] In some embodiments, the effector T cells and / or central memory T cells obtained from the third TIL population in step (c) exhibit increased CD8 and CD28 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population in step (b).

[0120] In some embodiments, the cancer is a solid tumor.

[0121]

[0121] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0122]

[0122] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is HNSCC. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is glioblastoma (including GBM). In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is a high-frequency mutated cancer. In some embodiments, the cancer is a pediatric high-frequency mutated cancer.

[0123]

[0123] In some embodiments, the container is a closed container. In some embodiments, the container is a G container. In some embodiments, the container is a GREX-10. In some embodiments, the closed container includes a GREX-100. In some embodiments, the closed container includes a GREX-500.

[0124]

[0124] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population produced by the method of any of the foregoing claims.

[0125]

[0125] In some embodiments, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from a patient's tumor tissue, and the therapeutic TIL population provides increased efficacy, increased interferon gamma production, and / or increased polyclonality.

[0126]

[0126] In some embodiments, it is the therapeutic TIL population described above and herein that provides increased interferon gamma production.

[0127]

[0127] In some embodiments, it is the therapeutic TIL population described above and herein that provides increased polyclonality.

[0128]

[0128] In some embodiments, it is the therapeutic TIL population described above and herein that provides increased efficacy.

[0129]

[0129] In some embodiments, it is the therapeutic TIL population described above and herein that is capable of producing at least one-fold more interferon gamma as compared to TIL prepared by a process longer than 16 days.

[0130]

[0130] In some embodiments, it is the therapeutic TIL population described above and herein that is capable of producing at least two-fold more interferon gamma as compared to TIL prepared by a process longer than 16 days.

[0131]

[0131] In some embodiments, it is the therapeutic TIL population described above and herein that is capable of producing at least three-fold more interferon gamma as compared to TIL prepared by a process longer than 16 days.

[0132]

[0132] 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 one-fold more interferon gamma as compared to TIL prepared by a process in which the first expansion culture of TIL is carried out without any added antigen-presenting cells (APCs).

[0133]

[0133] In some embodiments, the therapeutic TIL population described above and herein is capable of producing at least two-fold more interferon gamma as compared to TIL prepared by a process in which the first expansion culture of TIL is performed without any added APCs.

[0134]

[0134] In some embodiments, the therapeutic TIL population described above and herein is capable of producing at least three-fold more interferon gamma as compared to TIL prepared by a process in which the first expansion culture of TIL is performed without any added APCs.

[0135]

[0135] 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 one-fold more interferon gamma as compared to TIL prepared by a process in which the first expansion culture of TIL is performed without any added OKT3.

[0136]

[0136] In some embodiments, the therapeutic TIL population described above and herein is capable of producing at least two-fold more interferon gamma as compared to TIL prepared by a process in which the first expansion culture of TIL is performed without any added OKT3.

[0137]

[0137] In some embodiments, the therapeutic TIL population is capable of producing at least three-fold more interferon gamma as compared to TIL prepared by a process in which the first expansion culture of TIL is performed without any added OKT3.

[0138]

[0138] 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 one-fold more interferon-gamma as compared to TILs prepared by a process in which a first expansion culture of TILs is carried out without added antigen-presenting cells (APCs) and without added OKT3.

[0139]

[0139] In some embodiments, the therapeutic TIL population described above and herein is capable of producing at least two-fold more interferon-gamma as compared to TILs prepared by a process in which a first expansion culture of TILs is carried out without the addition of added antigen-presenting cells (APCs) and without added OKT3.

[0140]

[0140] In some embodiments, the therapeutic TIL population described above and herein is capable of producing at least three-fold more interferon-gamma as compared to TILs prepared by a process in which a first expansion culture of TILs is carried out without added antigen-presenting cells (APCs) and without added OKT3.

[0141]

[0141] In some embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described above and herein and a pharmaceutically acceptable carrier.

[0142]

[0142] In some embodiments, the present invention provides a sterile infusion bag containing a TIL composition as described above and herein.

[0143]

[0143] In some embodiments, the present invention provides a cryopreserved preparation of a therapeutic TIL population as described above and herein.

[0144]

[0144] In some embodiments, the present invention provides a tumor-infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described above and herein and a cryopreservation medium.

[0145]

[0145] In some embodiments, the cryopreservation medium is the TIL composition described above and herein that contains DMSO.

[0146]

[0146] In some embodiments, the cryopreservation medium is the TIL composition described above and herein that contains 7 - 10% DMSO.

[0147]

[0147] In some embodiments, the present invention provides a cryopreserved preparation of the TIL composition as described above and herein.

[0148]

[0148] In some embodiments, the tumor - infiltrating lymphocyte (TIL) composition described above and herein for use as a medicament.

[0149]

[0149] In some embodiments, the tumor - infiltrating lymphocyte (TIL) composition described above and herein for use in the treatment of cancer.

[0150]

[0150] In some embodiments, the tumor - infiltrating lymphocyte (TIL) composition described above and herein for use in the treatment of solid - tumor cancer.

[0151]

[0151] In some embodiments, the tumor - infiltrating lymphocyte (TIL) composition described above and herein for use in the treatment of cancer selected from melanoma, ovarian cancer, cervical cancer, non - small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0152]

[0152] In some embodiments, the tumor - infiltrating lymphocyte (TIL) composition described above and herein is for use in the treatment of cancer selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0153]

[0153] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is melanoma.

[0154]

[0154] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is HNSCC.

[0155]

[0155] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is cervical cancer.

[0156]

[0156] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is NSCLC.

[0157]

[0157] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is glioblastoma (including GBM).

[0158]

[0158] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is gastrointestinal cancer.

[0159]

[0159] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is high-frequency mutated cancer.

[0160]

[0160] In some embodiments, the TIL compositions described above and herein are for use in the treatment of cancer, which is pediatric high-frequency mutated cancer.

[0161]

[0161] In some embodiments, the present invention provides for the use of the TIL compositions described above and herein in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of a tumor infiltrating lymphocyte (TIL) composition.

[0162]

[0162] In some embodiments, the present invention provides for the use of a TIL composition as described above and herein, wherein the cancer is a solid tumor.

[0163]

[0163] In some embodiments, the present invention provides for the use of a TIL composition as described above and herein, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0164]

[0164] In some embodiments, the present invention provides for the use of the TIL composition described above and herein, wherein 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 present invention provides that the cancer is HNSCC. In some embodiments, the present invention provides that the cancer is cervical cancer. In some embodiments, the present invention provides that the cancer is NSCLC. In some embodiments, the present invention provides that the cancer is glioblastoma (including GBM). In some embodiments, the present invention provides that the cancer is gastrointestinal cancer. In some embodiments, the present invention provides that the cancer is a high-frequency mutant cancer. In some embodiments, the present invention provides that the cancer is a pediatric high-frequency mutant cancer.

[0165]

[0165] In some embodiments, the present invention provides the TIL compositions described above and herein for use in a method of treating a subject's cancer, the method comprising administering to the subject a therapeutically effective dose of a tumor-infiltrating lymphocyte (TIL) composition. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

[0166]

[0166] In some embodiments, the present invention provides a method of treating a subject's cancer, the method comprising administering to the subject a therapeutically effective dose of a tumor-infiltrating lymphocyte (TIL) composition as described above and herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

[0167]

[0167] In some embodiments, the cancer is selected from the group consisting of melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is HNSCC. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is glioblastoma (including GBM). In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is a high-frequency mutation cancer. In some embodiments, the cancer is a pediatric high-frequency mutation cancer.

[0168]

[0168] In some embodiments, the present invention provides a method for expanding T cells, comprising: (a) performing a first priming expansion culture of a first T cell population obtained from a donor by culturing the first T cell population to effect proliferation and effecting first priming activation of the first T cell population; (b) after activation of the first T cell population primed in step (a) begins to wane, performing a second rapid expansion culture of the first T cell population by culturing the first T cell population to effect proliferation and effecting boosting activation of the first T cell population to obtain a second T cell population; and (c) recovering the second T cell population.

[0169]

[0169] In some embodiments, the first priming expansion culture of step (a) is performed over a period of up to 7 days.

[0170]

[0170] In some embodiments, the second rapid expansion culture of step (b) is performed over a period of up to 11 days. In some embodiments, the second rapid expansion culture of step (b) is performed over a period of up to 9 days.

[0171]

[0171] In some embodiments, the first priming expansion culture of step (a) is performed over a period of 7 days and the second rapid expansion culture of step (b) is performed over a period of 9 days.

[0172]

[0172] In some embodiments, in step (a) of the methods described herein, the first T cell population is cultured in a first culture medium comprising OKT-3 and IL-2.

[0173]

[0173] In some embodiments, the first culture medium comprises OKT-3, IL-2, and antigen presenting cells (APCs).

[0174]

[0174] In some embodiments, in step (b) of the methods described herein, the first T cell population is cultured in a second culture medium containing OKT-3, IL-2, and antigen presenting cells (APCs).

[0175]

[0175] In some embodiments, the first T cell population in step (a) is cultured in a first culture medium in a container containing a first gas permeable surface, the first culture medium contains OKT-3, IL-2, and a first antigen presenting cell (APC) population, the first APC population is exogenous to the donor of the first T cell population, and the first APC population is laminated on the first gas permeable surface. In step (b), the first T cell population is cultured in a second culture medium in the container, the second culture medium contains OKT-3, IL-2, and a second APC population, the second APC population is exogenous to the donor of the first T cell population, and the second APC population is laminated on the first gas permeable surface, and the second APC population is larger than the first APC population.

[0176]

[0176] In some embodiments, the ratio of the number of APCs in the second APC population to the number of APCs in the first APC population is about 2:1.

[0177]

[0177] In some embodiments, the number of APCs in the first APC population is about 2.5×10 8 cells, and the number of APCs in the second APC population is about 5×10 8 cells.

[0178]

[0178] In some embodiments, in step (a) of the methods described herein, the first APC population is laminated on the first gas permeable surface with an average thickness of two layers of APCs.

[0179]

[0179] In some embodiments, in step (b) of the methods described herein, the second APC population is laminated on the first gas permeable surface with an average thickness in the range of 4 to 8 layers of APCs.

[0180]

[0180] In some embodiments, the ratio of the average number of layers of APCs deposited on the first gas-permeable surface in step (b) to the average number of layers of APCs deposited on the first gas-permeable surface in step (a) is 2:1.

[0181]

[0181] In some embodiments, the APC is peripheral blood mononuclear cells (PBMC).

[0182]

[0182] In some embodiments, the PBMCs are irradiated and exogenous to the donor of the first T cell population.

[0183]

[0183] In some embodiments, the T cells are tumor-infiltrating lymphocytes (TIL).

[0184]

[0184] In some embodiments, the T cells are marrow-infiltrating lymphocytes (MIL).

[0185]

[0185] In some embodiments, the T cells are peripheral blood lymphocytes (PBL).

[0186]

[0186] In any of the foregoing embodiments, the OKT-3 concentration in the first primary stimulation expansion culture is about 30 ng / mL, and the OKT-3 concentration in the second rapid expansion culture is about 30 ng / mL. In any of the foregoing embodiments, the OKT-3 concentration in the first primary stimulation expansion culture is about 30 ng / mL, and the OKT-3 concentration in the second rapid expansion culture is about 60 ng / mL.

Brief Description of the Drawings

[0187] Brief Description of the Drawings

Figure 1A

[0187] Shows a comparison of the 2A process (a process of about 22 days) and embodiments of the Gen3 process for TIL production (a process of about 14 to 16 days).

Figure 1B

[0187] Exemplary process Gen3 chart (process of about 14 to 16 days) providing an overview of steps A - F.

Figure 1C

[0187] Exemplary process Gen3 chart (process of about 14 to 16 days) providing an overview of steps A - F.

Figure 2

[0188] Provide an experimental flowchart for comparing GEN2 (process 2A) with GEN3.

Figure 3

[0189] Phenotypic characterization of TIL products in M1085T - Gen2 and Gen3 processes.

Figure 4

[0190] Memory marker analysis of TIL products from M1085T - Gen2 and Gen3 processes.

Figure 5A

[0191] L4054 activation and exhaustion markers, gated on CD4+.

Figure 5B

[0191] L4054 activation and exhaustion markers, gated on CD8+.

Figure 6A

[0192] L4055 activation and exhaustion markers, gated on CD4+.

Figure 6B

[0192] L4055 activation and exhaustion markers, gated on CD8+.

Figure 7A

[0193] IFNγ production (pg / mL): L4054 for Gen2 and Gen3 processes: Each bar represented here is the mean + SEM of IFNγ levels for stimulated, unstimulated, and media controls. Optical density measured at 450 nm.

Figure 7B

[0193] IFNγ production (pg / mL): L4055 for Gen2 and Gen3 processes: Each bar represented here is the mean + SEM of IFNγ levels for stimulated, unstimulated, and media controls. Optical density measured at 450 nm.

Figure 7C

[0193] IFNγ production (pg / mL): M1085T for Gen2 and Gen3 processes: Each bar represented here is the mean + SEM of IFNγ levels for stimulated, unstimulated, and media controls. Optical density measured at 450 nm.

Figure 8A

[0194] ELISA analysis of IL-2 concentration in cell culture supernatant: L4054. Each bar represented here is the mean value + SEM of IL-2 levels in the spent media. Optical density measured at 450 nm.

Figure 8B

[0194] ELISA analysis of IL-2 concentration in cell culture supernatant: L4055. Each bar represented here is the mean value + SEM of IL-2 levels in the spent media. Optical density measured at 450 nm.

Figure 9A

[0195] Quantification of glucose and lactate (g / L) in spent media: Glucose: A decrease in glucose was observed throughout the REP expansion culture for two tumor lines and both processes. Conversely, as expected, an increase in lactate was observed. Both the decrease in glucose and the increase in lactate were equivalent between the Gen2 and Gen3 processes.

Figure 9B

[0195] Quantification of glucose and lactate (g / L) in spent media: Lactate: A decrease in glucose was observed throughout the REP expansion culture for two tumor lines and both processes. Conversely, as expected, an increase in lactate was observed. Both the decrease in glucose and the increase in lactate were equivalent between the Gen2 and Gen3 processes.

Figure 10A

[0196] Quantification of L-glutamine in the spent media of L4054 and L4055.

Figure 10B

[0196] Quantification of GlutaMAX in the spent media of L4054 and L4055.

Figure 10C

[0196] Quantification of ammonia in the spent media for L4054 and L4055.

Figure 11

[0197] Telomere length analysis. The relative telomere length (RTL) value indicates the average telomere fluorescence per chromosome / genome in the Gen2 and Gen3 processes of telomere fluorescence per chromosome / genome of the control cell line (1301 leukemia cell line) using the DAKO kit.

Figure 12

[0198] Analysis of unique CDR3 sequences of the TIL final products of L4054 and L4055 in Gen2 and Gen3. The number of unique TCRB clone types identified from 1×106 cells collected on the recovery days of the Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14 - 16) is shown in columns. Gen3 shows higher clonal diversity compared to Gen2 based on the number of unique peptide CDRs within the sample.

Figure 13

[0199] Frequency of unique CDR3 sequences of the final cell products recovered with L4054 IL (Gen2 (e.g., day 22) and Gen3 process (e.g., days 14 - 16)).

Figure 14

[0200] Frequency of unique CDR3 sequences of the final cell products recovered with L4055 TIL (Gen2 (e.g., day 22) and Gen3 process (e.g., days 14 - 16)).

Figure 15

[0201] Diversity indices of the TIL final products of L4054 and L4055 in Gen2 and Gen3. The Shannon entropy diversity index is a more reliable and common indicator for comparison. Gen3 L4054 and L4055 showed slightly higher diversity than Gen2.

Figure 16

[0202] Raw data of the cell numbers at day 7 - start of Gen3 REP shown in Table 22 (see Example 5 below).

Figure 17

[0203] Raw data of the cell numbers at day 11 - start of Gen2 REP and Gen3 scale - up shown in Table 22 (see Example 5 below).

Figure 18

[0204] Raw data of cell counts on the 16th day - Gen2 scale-up and Gen3 recovery (e.g., on the 16th day) shown in Table 23 (see Example 5 below).

Figure 19

[0205] Raw data of cell counts on the 22nd day - Gen2 recovery (e.g., on the 22nd day) shown in Table 23 (see Example 5 below). In the case of L4054 Gen2, since it was the total number of studies, the number after LOVO was extrapolated to 4 flasks. One flask was contaminated and estimated to be a total of 67E+10.

Figure 20

[0206] Raw data of the flow cytometry results shown in Figures 3A, 4A, and 4B.

Figure 21

[0207] Raw data of the flow cytometry results shown in Figures 3C and 4C.

Figure 22

[0208] Raw data of the flow cytometry results shown in Figures 5 and 6.

Figure 23A

[0209] Raw data of the IFNγ production assay results of the L4054 sample shown in Figure 7.

Figure 23B

[0209] Raw data of the IFNγ production assay results of the L4054 sample shown in Figure 7.

Figure 24A

[0210] Raw data of the IFNγ production assay results of the L4055 sample shown in Figure 7.

Figure 24B

[0210] Raw data of the IFNγ production assay results of the L4055 sample shown in Figure 7.

Figure 25A

[0211] Raw data of the IFNγ production assay results of the M1085T sample shown in Figure 7.

Figure 25B

[0211] Raw data of the IFNγ production assay results of the M1085T sample shown in Figure 7.

Figure 26A

[0212] Raw data of the IL-2 ELISA assay results shown in Figure 8.

Figure 26B

[0212] Raw data of the IL-2 ELISA assay results shown in FIG. 8.

Figure 27

[0213] Raw data of the metabolic substrate and metabolic analysis results shown in FIGS. 9 and 10.

Figure 28

[0214] Raw data of the relative telomere length analysis results shown in FIG. 11.

Figure 29

[0215] Raw data of the unique CD3 sequence and clonal diversity analysis results shown in FIGS. 12 and 15.

Figure 30

[0216] Shows a comparison of various embodiments of different Gen2 (2A process) and Gen3.1 processes.

Figure 31

[0217] Table explaining various characteristics of embodiments of Gen2, Gen2.1 and Gen3.0 processes.

Figure 32

[0218] Overview of the culture medium conditions of an embodiment of the Gen3 process, referred to as Gen3.1.

Figure 33

[0219] Table explaining various characteristics of embodiments of Gen2, Gen2.1 and Gen3.0 processes.

Figure 34

[0220] Table comparing various characteristics of embodiments of Gen2 and Gen3.0 processes.

Figure 35

[0221] Table providing the use of the culture medium in various embodiments of the described expansion culture process.

Figure 36

[0222] Phenotype comparison: Embodiments of Gen3.0 and Gen3.1 of the process showed equivalent CD28, CD27 and CD57 expression.

Figure 37

[0223] Higher production of IFNγ in the final product of Gen3. To compare both processes, IFNγ analysis (by ELISA) was evaluated in frozen culture supernatants. Fresh TIL products were used at each Gen2 (e.g., day 22) and Gen3 process (e.g., day 16), and for each tumor, it was an overnight stimulation with coated anti-CD3 plates. Each bar represented here are the IFNγ levels of stimulated, non-stimulated and medium controls. Shown as L4055.

Figure 38A

[0224] Unique CDR3 sequence analysis of TIL final products: The number of unique TCR B clone types identified from 1×106 cells collected at Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14 - 16) are shown in columns. Gen3 shows higher clonal diversity compared to Gen2 based on the number of unique peptide CDRs within the sample.

Figure 38B

[0224] Diversity index of TIL final products: The Shannon entropy diversity index is a more reliable general indicator for comparison. Gen3 showed slightly higher diversity than Gen2.

Figure 39

[0225] 199 sequences are shared between the final products of Gen3 and Gen2 and correspond to 97.07% of the top 80% of unique CDR3 sequences from Gen2 that are shared with the final product of Gen3.

Figure 40

[0226] 1833 sequences are shared between the final products of Gen3 and Gen2 and correspond to 99.45% of the top 80% of unique CDR3 sequences from Gen2 that are shared with the final product of Gen3.

Figure 41

[0227] Schematic diagram of an exemplary embodiment of the Gen3 process (16-day process).

Figure 42

[0228] Schematic diagram of an exemplary embodiment for expanding TIL from hematopoietic tumors using the Gen3 process. On day 0, a T cell fraction (CD3+, CD45+) is separated from apheresis products rich in lymphocytes, whole blood, or tumor digest (fresh or thawed) using a positive or negative selection method, i.e., removal of T cells using T cell markers (such as CD2, CD3, etc., or removal of other cells leaving T cells) or gradient centrifugation.

Figure 43

[0229] Schematic diagram of an exemplary embodiment for comparing Process 1C to Process 2A and expanding TIL according to Process 2A.

Figure 44A

[0230] Schematic diagrams of different versions for expanding TIL according to the minilaparotomy process. (A) represents Version 1, and the expansion culture process is approximately 21 - 33 days from Steps A - E.

Figure 44B

[0230] Schematic diagrams of different versions for expanding TIL according to the minilaparotomy process. (B) represents Version 2, and the expansion culture process is approximately 17 - 24 days from Steps A - E.

Figure 44C

[0230] Schematic diagrams of different versions for expanding TIL according to the minilaparotomy process. (C) represents a comparison with Process 2A of Versions 1 and 2.

Figure 45

[0231] Showing the pathological information of lymphoma tumors.

Figure 46

[0232] Showing a comparison of different subsets of lymphoma and melanoma TIL, indicating that the effector memory (EM) subset of lymphoma TIL is significantly higher than that of melanoma TIL. Showing the CD8+ subset of TEMRA.

Figure 47

[0233] Showing a comparison of different subsets of lymphoma and melanoma TIL, indicating that the CD28+CD4+ subset of lymphoma TIL is significantly higher than these subsets of melanoma TIL. Showing the CD28+CD8+ subset.

Figure 48

[0234] Comparison of CD4+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs, which shows differentiation markers. The red line in the graph represents the median. CM refers to central memory T cells, EM refers to effector memory T cells, and TEMRA refers to effector memory CD45RA+ T cells.

Figure 49

[0235] Comparison of CD8+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs, which shows differentiation markers. The red line in the graph represents the median. CM refers to central memory T cells, EM refers to effector memory T cells, and TEMRA refers to effector memory CD45RA+ T cells.

Figure 50

[0236] Comparison of CD4+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs, which shows exhaustion markers. The red line in the graph represents the median. LAG3 refers to lymphocyte activation gene 3, PD1 refers to programmed death 1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains.

Figure 51

[0237] Comparison of CD8+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs, which shows exhaustion markers. The red line in the graph represents the median. LAG3 refers to lymphocyte activation gene 3, PD1 refers to programmed death 1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains.

Figure 52

[0238] Comparison of cell types between non-Hodgkin lymphoma TILs and melanoma TILs is shown. NK refers to natural killer cells, and TCRab refers to cells expressing T cell receptors with alpha and beta chains.

Figure 53

[0239] Results of the bioluminescence redirected lysis assay (BRLA) are shown.

Figure 54

[0240] Results of the interferon-γ (IFN-γ) enzyme-linked immunosorbent assay (ELISA) of lymphoma TILs versus melanoma TILs are shown.

Figure 55

[0241] Shows the results of the enzyme-linked immunosorbent spot (ELIspot) assay for lymphoma TILs.

Figure 56

[0242] Shows the ELIspot assay results for melanoma TILs.

Figure 57

[0243] Shows the results of NANOSTRING NCOUNTER analysis, which indicates that lymphoma TILs express higher levels of RORC IL17A (TH17 phenotype) and GATA3 (Th2 phenotype) compared to melanoma TILs. Each gene is highlighted by the red frame in the heatmap.

Figure 58

[0244] Shows structures I-A and I-B of the 4-1BB agonistic fusion protein. The cylinders refer to individual polypeptide binding domains. Structures I-A and I-B contain three linearly linked TNFRSF binding domains derived from, for example, an antibody that binds to 4-1BBL or 4-1BB, which are folded to form a trivalent protein, and then linked to a second trivalent protein via IgG1-Fc (including CH3 and CH2 domains), and then this is used to link the two trivalent proteins via disulfide bonds (small elongated ellipses), stabilizing the structure and providing an agonist that can form a signal transduction complex with the intracellular signal transduction domains and signal transduction proteins of six receptors. The TNFRSF binding domains shown as cylinders can be scFv domains containing, for example, VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility and Glu and Lys for solubility.

[0188] Brief description of the sequence listing

[0245] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0189]

[0246] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0190]

[0247] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0191]

[0248] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0192]

[0249] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0193]

[0250] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0194]

[0251] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0195]

[0252] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0196]

[0253] SEQ ID NO: 9 is the amino acid sequence of human 4-1BB.

[0197]

[0254] SEQ ID NO: 10 is the amino acid sequence of mouse 4-1BB.

[0198]

[0255] SEQ ID NO: 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0199]

[0256] SEQ ID NO: 12 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0200]

[0257] SEQ ID NO: 13 is the variable heavy chain (VH) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0201]

[0258] SEQ ID NO: 14 is the variable light chain (VL) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0202]

[0259] Sequence number 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0203]

[0260] Sequence number 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0204]

[0261] Sequence number 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0205]

[0262] Sequence number 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0206]

[0263] Sequence number 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0207]

[0264] Sequence number 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0208]

[0265] Sequence number 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0209]

[0266] Sequence number 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0210]

[0267] Sequence number 23 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0211]

[0268] Sequence number 24 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0212]

[0269] SEQ ID NO: 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0213]

[0270] SEQ ID NO: 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0214]

[0271] SEQ ID NO: 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0215]

[0272] SEQ ID NO: 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0216]

[0273] SEQ ID NO: 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0217]

[0274] SEQ ID NO: 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0218]

[0275] SEQ ID NO: 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0219]

[0276] SEQ ID NO: 32 is the linker of the TNFRSF agonist fusion protein.

[0220]

[0277] SEQ ID NO: 33 is the linker of the TNFRSF agonist fusion protein.

[0221]

[0278] SEQ ID NO: 34 is the linker of the TNFRSF agonist fusion protein.

[0222]

[0279] SEQ ID NO: 35 is the linker of the TNFRSF agonist fusion protein.

[0223]

[0280] SEQ ID NO: 36 is the linker of the TNFRSF agonist fusion protein.

[0224]

[0281] SEQ ID NO: 37 is the linker of the TNFRSF agonist fusion protein.

[0225]

[0282] SEQ ID NO: 38 is the linker of the TNFRSF agonist fusion protein.

[0226]

[0283] SEQ ID NO: 39 is the linker of the TNFRSF agonist fusion protein.

[0227]

[0284] SEQ ID NO: 40 is the linker of the TNFRSF agonist fusion protein.

[0228]

[0285] SEQ ID NO: 41 is the linker of the TNFRSF agonist fusion protein.

[0229]

[0286] SEQ ID NO: 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0230]

[0287] SEQ ID NO: 43 is the linker of the TNFRSF agonist fusion protein.

[0231]

[0288] SEQ ID NO: 44 is the linker of the TNFRSF agonist fusion protein.

[0232]

[0289] SEQ ID NO: 45 is the linker of the TNFRSF agonist fusion protein.

[0233]

[0290] SEQ ID NO: 46 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0234]

[0291] SEQ ID NO: 47 is the soluble portion of the 4-1BBL polypeptide.

[0235]

[0292] SEQ ID NO: 48 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0236]

[0293] SEQ ID NO: 49 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 1.

[0237]

[0294] SEQ ID NO: 50 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0238]

[0295] SEQ ID NO: 51 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0239]

[0296] SEQ ID NO: 52 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E3-2.

[0240]

[0297] SEQ ID NO: 53 is the light chain variable region (VL) of the 4-1BB agonist antibody H39E3-2.

[0241]

[0298] SEQ ID NO: 54 is the amino acid sequence of human OX40.

[0242]

[0299] SEQ ID NO: 55 is the amino acid sequence of mouse OX40.

[0243]

[0300] SEQ ID NO: 56 is the heavy chain of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0244]

[0301] SEQ ID NO: 57 is the light chain of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0245]

[0302] SEQ ID NO: 58 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0246]

[0303] Accession number 59 is the variable light chain (VL) of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0247]

[0304] Accession number 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0248]

[0305] Accession number 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0249]

[0306] Accession number 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0250]

[0307] Accession number 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0251]

[0308] Accession number 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0252]

[0309] Accession number 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabalixizumab (MEDI-0562).

[0253]

[0310] Accession number 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0254]

[0311] Accession number 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0255]

[0312] Accession number 68 is the variable heavy chain (VH) of the OX40 agonist monoclonal antibody 11D4.

[0256]

[0313] SEQ ID NO: 69 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 11D4.

[0257]

[0314] SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0258]

[0315] SEQ ID NO: 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0259]

[0316] SEQ ID NO: 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0260]

[0317] SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0261]

[0318] SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0262]

[0319] SEQ ID NO: 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0263]

[0320] SEQ ID NO: 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0264]

[0321] SEQ ID NO: 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0265]

[0322] SEQ ID NO: 78 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 18D8.

[0266]

[0323] SEQ ID NO: 79 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 18D8.

[0267]

[0324] The sequence number 80 is the heavy-chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0268]

[0325] The sequence number 81 is the heavy-chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0269]

[0326] The sequence number 82 is the heavy-chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0270]

[0327] The sequence number 83 is the light-chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0271]

[0328] The sequence number 84 is the light-chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0272]

[0329] The sequence number 85 is the light-chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0273]

[0330] The sequence number 86 is the heavy-chain variable region (VH) of the OX40 agonist monoclonal antibody Hu119-122.

[0274]

[0331] The sequence number 87 is the light-chain variable region (VL) of the OX40 agonist monoclonal antibody Hu119-122.

[0275]

[0332] The sequence number 88 is the heavy-chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0276]

[0333] The sequence number 89 is the heavy-chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0277]

[0334] The sequence number 90 is the heavy-chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0278]

[0335] Sequence number 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0279]

[0336] Sequence number 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0280]

[0337] Sequence number 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0281]

[0338] Sequence number 94 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu106-222.

[0282]

[0339] Sequence number 95 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu106-222.

[0283]

[0340] Sequence number 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0284]

[0341] Sequence number 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0285]

[0342] Sequence number 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0286]

[0343] Sequence number 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0287]

[0344] Sequence number 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0288]

[0345] Sequence number 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0289]

[0346] SEQ ID NO: 102 is the amino acid sequence of OX40 ligand (OX40L).

[0290]

[0347] SEQ ID NO: 103 is the soluble portion of the OX40L polypeptide.

[0291]

[0348] SEQ ID NO: 104 is an alternative soluble portion of the OX40L polypeptide.

[0292]

[0349] SEQ ID NO: 105 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 008.

[0293]

[0350] SEQ ID NO: 106 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 008.

[0294]

[0351] SEQ ID NO: 107 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 011.

[0295]

[0352] SEQ ID NO: 108 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 011.

[0296]

[0353] SEQ ID NO: 109 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 021.

[0297]

[0354] SEQ ID NO: 110 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 021.

[0298]

[0355] SEQ ID NO: 111 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 023.

[0299]

[0356] SEQ ID NO: 112 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 023.

[0300]

[0357] SEQ ID NO: 113 is the variable heavy chain region (VH) of an OX40 agonist monoclonal antibody.

[0301]

[0358] SEQ ID NO: 114 is the variable light chain region (VL) of an OX40 agonist monoclonal antibody.

[0302]

[0359] SEQ ID NO: 115 is the variable heavy chain region (VH) of an OX40 agonist monoclonal antibody.

[0303]

[0360] SEQ ID NO: 116 is the variable light chain region (VL) of an OX40 agonist monoclonal antibody.

[0304]

[0361] SEQ ID NO: 117 is the variable heavy chain region (VH) of a humanized OX40 agonist monoclonal antibody.

[0305]

[0362] SEQ ID NO: 118 is the variable heavy chain region (VH) of a humanized OX40 agonist monoclonal antibody.

[0306]

[0363] SEQ ID NO: 119 is the variable light chain region (VL) of a humanized OX40 agonist monoclonal antibody.

[0307]

[0364] SEQ ID NO: 120 is the variable light chain region (VL) of a humanized OX40 agonist monoclonal antibody.

[0308]

[0365] SEQ ID NO: 121 is the variable heavy chain region (VH) of a humanized OX40 agonist monoclonal antibody.

[0309]

[0366] SEQ ID NO: 122 is the variable heavy chain region (VH) of a humanized OX40 agonist monoclonal antibody.

[0310]

[0367] SEQ ID NO: 123 is the variable light chain region (VL) of a humanized OX40 agonist monoclonal antibody.

[0311]

[0368] SEQ ID NO: 124 is the variable light chain region (VL) of a humanized OX40 agonist monoclonal antibody.

[0312]

[0369] SEQ ID NO: 125 is the variable heavy chain region (VH) of an OX40 agonist monoclonal antibody.

[0313]

[0370] SEQ ID NO: 126 is the variable light chain region (VL) of an OX40 agonist monoclonal antibody.

[0314]

[0371] SEQ ID NO: 127 is a partial sequence of human IL-2Rβ, residues 1 to 235.

[0315]

[0372] SEQ ID NO: 128 is a partial sequence of mouse IL-2Rβ, residues 1 to 238.

[0316]

[0373] SEQ ID NO: 129 is mouse IL-2.

[0317]

[0374] SEQ ID NO: 130 is human IL-2.

[0318]

[0375] SEQ ID NO: 131 is an example of orthogonal human IL-2.

[0319]

[0376] SEQ ID NO: 132 is an example of orthogonal human IL-2.

[0320]

[0377] SEQ ID NO: 133 is an example of orthogonal human IL-2.

[0321]

[0378] SEQ ID NO: 134 is an example of orthogonal human IL-2.

[0322]

[0379] SEQ ID NO: 135 is the human IL2-Rβ subunit.

[0323]

[0380] SEQ ID NOs: 136 to 152 are PCR primers useful for generating a site-specific mutant IL-2 library.

[0324]

[0381] SEQ ID NO: 153 is the human IL2-Rβ subunit.

[0325]

[0382] Accession No. 154 Human IL2-Rα subunit.

[0326]

[0383] Accession No. 155 Human IL2-Rγ subunit.

[0327]

[0384] Accession No. 156 Human IL-2.

Mode for Carrying Out the Invention

[0328] Detailed Description of the Invention I. Definitions

[0385] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are hereby incorporated by reference in their entirety.

[0329]

[0386] The term "in vivo" refers to events occurring within the body of a subject.

[0330]

[0387] The term "in vitro" refers to events occurring outside the body of a subject. In vitro assays include cell-based assays using live or dead cells, and may also include cell-free assays that do not utilize intact cells.

[0331]

[0388] The term "ex vivo" refers to events involving the treatment or performance of a treatment on cells, tissues, and / or organs removed from the body of a subject. Appropriately, the cells, tissues, and / or organs are returned to the body of the subject by surgery or a treatment method.

[0332]

[0389] The term "rapid expansion culture" means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold) over a period of one week, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold or 90-fold) over a period of one week or most preferably at least about 100-fold over a period of one week. Multiple rapid expansion culture protocols are outlined below.

[0333]

[0390] As used herein, "tumor infiltrating lymphocytes" or "TIL" means a population of cells that have migrated from the bloodstream of a subject into a tumor and were originally obtained as white blood cells. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly obtained" or "freshly harvested"), and "secondary TILs" are any population of TIL cells that have been expanded or grown as discussed herein, including but not limited to bulk TILs and expanded culture TILs ("REP TILs" or "post-REP TILs"). TIL cell populations can include genetically modified TILs.

[0334]

[0391] As used herein, "cell population" (including TILs) means a large number of cells that share a common trait. Generally, the population is generally in the range of 1×10 6 ~ 1×10 10 cells, and different TIL populations contain different numbers. For example, the initial growth of primary TILs in the presence of IL-2 results in a bulk TIL population of approximately 1×10 8 cells. REP expansion culture is generally performed such that a cell population for injection of 1.5×10 9 ~ 1.5×10 10 cells is provided. In some embodiments, the REP expansion culture is from 2.3×10 10 ~ 13.7×10 10It is performed to provide a population.

[0335]

[0392] As used herein, "cryopreserved TIL" means that TIL, whether primary, bulk or expanded culture (REP TIL), is processed and stored in the range of about -150°C to -60°C. General cryopreservation methods are described in other parts of this specification, including the examples. For clarity, "cryopreserved TIL" is distinguishable from cryopreserved tissue samples that can be used as a source of primary TIL.

[0336]

[0393] As used herein, "thawed cryopreserved TIL" means a population of TIL that was once cryopreserved but was then returned to a temperature above room temperature, including but not limited to cell culture temperature or a temperature at which the TIL can be administered to a patient, by a process.

[0337]

[0394] TIL can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and affect treatment. TIL can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TIL can be defined functionally by its ability to infiltrate solid tumors upon reintroduction into a patient.

[0338]

[0395] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such media may include media containing 7% - 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. The CS10 medium may be referred to by the trade name "CryoStor® CS10". The CS10 medium is a serum-free, animal component-free medium containing DMSO.

[0339]

[0396] The term "central memory T cells" refers to a subset of T cells that are CD45R0+ in humans and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells mainly secrete IL-2 and CD40L as effector molecules after TCR stimulation. Central memory T cells are dominant in the CD4 compartment of the blood and are proportionally concentrated in lymph nodes and tonsils in humans.

[0340]

[0397] The term "effector memory T cell" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but lack constitutive expression of CCR7 (CCR7lo) and have heterogeneous or low expression of CD62L (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factor of central memory T cells includes BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, after antigen stimulation. Effector memory T cells are predominant in the CD8 compartment of the blood and are proportionally concentrated in the lung, liver, and intestine in humans. CD8+ effector memory T cells have a large amount of perforin.

[0341]

[0398] The term "closed system" refers to a system that is closed to the external environment. In the method of the present invention, any closed system suitable for cell culture methods can be used. Examples of closed systems include, but are not limited to, closed-type G containers. After a tumor segment is added to the closed system, the system is not opened to the external environment until immediately before administration of the TIL to the patient.

[0342]

[0399] As used herein to describe the process of fragmenting a tumor, the terms "fragmentation", "fragment", and "fragmented" include mechanical fragmentation methods such as the fragmentation, slicing, dividing, and mincing of tumor tissue and any other method that disrupts the physical structure of tumor tissue.

[0343]

[0400] The terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as an antigen-presenting cell (PBMC is a type of antigen-presenting cell), the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0344]

[0401] The terms "peripheral blood lymphocytes" and "PBL" refer to T cells expanded from peripheral blood. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection of a T cell phenotype, such as a CD3+CD45+ T cell phenotype.

[0345]

[0402] The term "anti-CD3 antibody" refers to an antibody or variant thereof, such as a monoclonal antibody, that is a human, humanized, chimeric, or mouse antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otrexup, teprotumumab, and visilizumab.

[0346]

[0403] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof that includes a human, humanized, chimeric, or mouse antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells, including OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants thereof, including commercially available forms such as conservative amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 have been deposited with the American Type Culture Collection and have been assigned the ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 have also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and have been assigned the catalog number 86022706.

[0347]

Table 1

[0348]

[0404] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial) and recombinant IL-2 forms commercially 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 vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) 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 shown in Table 2 (SEQ ID NO: 4). The term IL-2 also includes pegylated forms of IL-2, including the pegylated IL2 prodrug NKTR-214 available from Nektar Therapeutics, South San Francisco, CA, USA, as described herein. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. 2014 / 0328791 A1 and International Publication No. 2012 / 065086 A1 (the disclosures of which are incorporated herein by reference). Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference.Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0349]

[0405] Orthogonal IL-2 is administered in the same units as wild-type IL-2. When orthogonal IL-2 is substituted for wild-type IL-2, the same dosage is used with respect to the international unit (IU) of orthogonal IL-2.

[0350]

Table 2

[0351]

[0406] The term "IL-2R" refers to the heterotrimeric IL-2 cytokine receptor. IL-2R includes α (alpha) (also called IL-2Rα, CD25 or Tac antigen), β (beta) (also called IL-2Rβ or CD122), and γ (gamma) (also called IL-2Rγ, γc, common gamma chain or CD132); these subunits are also part of the receptors for other cytokines. The mature (i.e., signal peptide cleaved) 525 amino acid sequence of human IL-2Rβ is shown in the following table. The standard sequences of human IL-2Rα and human IL-2Rγ are also shown.

[0352]

Table 3

[0353]

[0407] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. When activated by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell expansion and class II MHC expression and induces class switching to IgE and IgG1 expression from B cells. Recombinant human IL-4 suitable for use in the present invention is commercially available from a number of suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0354]

[0408] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin 7, which can be obtained from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, in a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0355]

[0409] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and their variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0356]

[0410] The term "IL-21" (also referred to herein as "IL21") refers to a pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is produced primarily by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0357]

[0411] When an "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention can be determined by a physician taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). Generally, a pharmaceutical composition containing tumor-infiltrating lymphocytes (e.g., secondary TIL or genetically modified cytotoxic lymphocytes) described herein is 10 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 、10 5 ~10 10 、10 5 ~10 11 、10 6 ~10 10 、10 6 ~10 11 、10 7 ~1011 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 or 10 9 ~10 10 It can be said that they can be administered at a dose of (including all integer values within these ranges) cells / kg body weight. The tumor-infiltrating lymphocyte composition (including, in some cases, genetically modified cytotoxic lymphocytes) can also be administered multiple times at these doses. Tumor-infiltrating lymphocytes (including, in some cases, genetically modified cytotoxic lymphocytes) can be administered by using infusion techniques generally known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0358]

[0412] The terms "hematological malignancy", "hematologic malignancy" or terms of correlated meaning refer to mammalian cancers and tumors of the hematopoietic and lymphatic tissues, including but not limited to blood, bone marrow, lymph nodes and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors". Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma and non-Hodgkin lymphoma. The term "B-cell hematological malignancy" refers to hematological malignancies that affect B cells.

[0359]

[0413] The term "solid tumor" refers to an abnormal mass of tissue that typically does not contain cysts or fluid regions. Solid tumors can be either benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Examples of 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 containing soft tissue (cancer cells) and supporting stromal cells in which the cancer cells are dispersed and which can provide a supportive microenvironment.

[0360]

[0414] The term "liquid tumor" refers to an abnormal mass of cells that is inherently fluid. Examples of liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other blood malignancies. TILs obtained from liquid tumors are also referred to herein as marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors that include liquid tumors circulating in peripheral blood can also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only by the tissue type from which the cells are derived.

[0361]

[0415] The term "microenvironment" as used herein can refer to the solid or blood tumor microenvironment as a whole or an individual subset of cells within the microenvironment. The tumor microenvironment as used herein refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from the host immune system, foster therapeutic resistance, and provide a niche for successful metastatic seeding," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that should be recognized by T cells, but elimination of the tumor by the immune system is rare due to immune suppression by the microenvironment.

[0362]

[0416] In one embodiment, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TILs according to the present invention. In some embodiments, a TIL population may be provided, wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TILs according to the present invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide at 60 mg / kg / day for 2 days (27 days and 26 days prior to TIL infusion) and fludarabine at 25 mg / m 2 / day for 5 days (27 - 23 days prior to TIL infusion). In one embodiment, after non-myeloablative chemotherapy and infusion of rTILs according to the present invention (day 0), the patient receives intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours up to physiological tolerance.

[0363]

[0417] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sink"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") for the patient prior to introduction of the rTILs of the present invention.

[0364]

[0418] As used herein, the terms "co-administer", "co-administering", "administered in combination with", "administering in combination with", "simultaneous" and "concurrent" refer to the administration to a subject of two or more active pharmaceutical ingredients (in a preferred embodiment of the present invention, for example, at least one potassium channel agonist in combination with a plurality of TILs) such that both the active pharmaceutical ingredients and / or their metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0365]

[0419] The terms "effective amount" or "therapeutically effective amount" refer to an amount of a compound or combination of compounds described herein that is sufficient to achieve the intended use, including, but not limited to, treating a disease. The therapeutically effective amount can vary depending on the intended use (in vitro or in vivo), the subject being treated and the disease state (e.g., the weight, age and sex of the subject), the severity of the disease state or the method of administration. The term also applies to the dosage that induces a specific response (e.g., a decrease in platelet adhesion and / or cell migration) in target cells. The specific dosage will vary depending on the specific compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered and the physical delivery system by which the compound is carried.

[0366]

[0420] The terms "treating", "being treated", "treatment" and the like refer to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms and / or therapeutic in terms of partially or completely curing a disease and / or its adverse effects caused by the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or alleviating one or more symptoms of the disease. "Treatment" is also intended to encompass the delivery of an agent for providing a pharmacological effect even in the absence of a disease or disorder. For example, "treatment" includes the delivery of a composition that can induce an immune response or confer immunity in the absence of a disease state, such as in the case of a vaccine.

[0367]

[0421] The term "heterologous" when used with respect to a nucleic acid or protein moiety indicates that the nucleic acid or protein contains two or more sub-sequences that are not found in nature in the same relationship to each other. For example, a nucleic acid is typically recombinantly produced and has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source or coding regions from different sources. Similarly, a heterologous protein indicates that the protein contains two or more sub-sequences that are not found in nature in the same relationship to each other (e.g., a fusion protein).

[0368]

[0422] The terms "sequence identity", "percent identity", and "sequence percent identity" (or synonyms thereof, such as "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence without considering conservative amino acid substitutions as part of sequence identity (introducing gaps if necessary). Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art for obtaining an alignment of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST programs available from the BLAST website of the National Center for Biotechnology Information of the United States government. The comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR are additional publicly available software programs that can be used to align sequences. One of ordinary skill in the art can determine appropriate parameters for maximum alignment by a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0369]

[0423] As used herein, the term "variant" encompasses, but is not limited to, a protein, antibody or fusion protein having an amino acid sequence that differs from the amino acid sequence of a reference antibody, protein or fusion protein by one or more substitutions, deletions and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody, protein or fusion protein. A variant may contain one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of the reference antibody, protein or fusion protein. Conservative substitutions can include, for example, substitutions of amino acids that are similarly charged or uncharged. A variant retains the ability to specifically bind to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

[0370]

[0424] As used herein, "tumor infiltrating lymphocytes" or "TIL" means a population of cells that have migrated from the bloodstream of a subject into a tumor and were originally obtained as white blood cells. TIL 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 include both primary and secondary TIL. "Primary TIL" are those obtained from patient tissue samples as outlined herein (and may be referred to as "freshly obtained" or "freshly harvested"), and "secondary TIL" are any population of TIL cells that have been expanded or grown as outlined herein, including but not limited to bulk TIL, expanded culture TIL ("REP TIL") and "reREP TIL" as considered herein. reREP TIL can include, for example, a second expanded culture TIL or a second additional expanded culture TIL (such as those described in step D of FIG. 1 that include TIL referred to as reREP TIL).

[0371]

[0425] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and affect treatment. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be defined functionally by their ability to infiltrate solid tumors upon reintroduction into the patient. TILs can be further characterized by potency - for example, TILs can be considered potent if interferon (IFN) release is higher than about 50 pg / mL, higher than about 100 pg / mL, higher than about 150 pg / mL, or higher than about 200 pg / mL.

[0372]

[0426] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" shall include any and all solvents, dispersing agents, 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. Their use in the therapeutic compositions of the present invention is contemplated, except where any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0373]

[0427] "Ortholog", "ortholog cytokine / receptor pair", "orthogonal cytokine / receptor pair", or "engineered cytokine / receptor pair" refers to a pair of genetically engineered proteins that are modified by (a) lack of binding to a native cytokine or cognate receptor; and (b) amino acid changes that specifically bind to a corresponding engineered (orthogonal) ligand or receptor. When bound, the orthogonal receptor activates signal transduction transmitted through native cellular elements to provide a biological activity that mimics its native response, which is specific to the engineered cell expressing the orthogonal receptor. The orthogonal receptor does not bind to endogenous corresponding cytokines, including the native counterparts of the orthogonal cytokine, while the orthogonal cytokine does not bind to any endogenous receptor, including the native counterpart of the orthogonal receptor. In some embodiments, the affinity of the orthogonal cytokine for the orthogonal receptor is equivalent to the affinity of the native cytokine for the native receptor, e.g., having an affinity that is at least about 1% of the affinity of the native cytokine-receptor pair, and can be at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100% and higher (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more) of the affinity of the native cytokine for the native receptor.

[0374]

[0428] As used herein, "does not bind" or "is unable to bind" refers to the absence of detectable binding or a binding that is not significant, i.e., has a binding affinity much lower than the binding affinity of the native ligand. Affinity can be determined by a competitive binding experiment that measures the binding of the receptor to a labeled ligand in the presence of various concentrations of unlabeled ligand. Typically, the concentration of the unlabeled ligand varies over a range that exceeds at least six orders of magnitude. Through the competitive binding experiment, IC 50 can be determined. As used herein, "IC 50 " refers to the concentration of unlabeled ligand required to inhibit 50% of the association between the receptor and the labeled ligand. IC 50 is an indicator of ligand-receptor binding affinity. A low IC 50represents high affinity and high IC 50 represents low affinity.

[0375]

[0429] The terms "about" and "approximately" refer to within a statistically meaningful range of values. Such range can be within one digit of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The allowable variation included in the term "about" or "approximately" depends on the particular system under study and is readily understandable to those skilled in the art. Further, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not exact and need not be exact, but may be approximate and / or larger or smaller, and as appropriate, reflect tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art. Generally, dimensions, sizes, formulations, parameters, shapes or other quantities or characteristics are "about" or "approximately" whether or not expressly stated to be so. Note that embodiments of widely different sizes, shapes and dimensions may refer to the terms described.

[0376]

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

[0377] II. TIL Manufacturing Process

[0431] While not limited to any particular theory, the first priming expansion culture that initially stimulates T cell activation and the subsequent second rapid expansion culture that boosts T cell activation as described in the method of the present invention are thought to enable the preparation of expanded T cells that retain a "more youthful" phenotype. Thus, the expanded T cells of the present invention are expected to exhibit greater cytotoxicity against cancer cells than T cells expanded by other methods. In particular, as taught by the method of the present invention, T cell activation that is initially stimulated by an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally exposure to antigen-presenting cells (APCs), and then boosted by subsequent exposure to additional anti-CD-3 antibody (e.g., OKT-3), IL-2, and APCs, restricts or avoids the maturation of T cells in culture, generating a population of T cells with a less mature phenotype that are thought to be less depleted by expansion in culture and exhibit higher cytotoxicity against cancer cells. In some embodiments, the step of the second rapid expansion culture is carried out by (a) culturing the T cells in a small-scale culture in a first container, e.g., a G-REX 100MCS container, for a period of about 3 to 4 days to perform the second rapid expansion culture, and then (b) transferring the T cells in the small-scale culture to a second container larger than the first container, e.g., a G-REX 500MCS container, and culturing the T cells from the small-scale culture in a larger-scale culture in the second container for a period of about 4 to 7 days, which is divided into multiple steps to achieve scale-up of the culture.In some embodiments, the rapid expansion culture step comprises: (a) performing a second rapid expansion culture by culturing T cells in a first vessel, such as a G-REX 100MCS container, in a first small-scale culture for a period of about 3 to 4 days, and then (b) transferring and distributing the T cells from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels that are the same size as the first vessel, wherein in each second vessel, a portion of the T cells from the first small-scale culture transferred to such second vessel is cultured in a second small-scale culture for a period of about 4 to 7 days, and is divided into multiple steps to achieve scale-out of the culture. In some embodiments, the rapid expansion culture step comprises: (a) performing a second rapid expansion culture by culturing T cells in a first vessel, such as a G-REX 100MCS container, in a small-scale culture for a period of about 3 to 4 days, and then (b) transferring and distributing the 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 vessels that are larger in size than the first vessel (e.g., a G-REX 500MCS container), wherein in each second vessel, a portion of the T cells from the small-scale culture transferred to such second vessel is cultured in a larger-scale culture for a period of about 4 to 7 days, and is divided into multiple steps to achieve scale-out and scale-up of the culture.In some embodiments, the rapid expansion culture step is to perform a second rapid expansion culture by culturing T cells in a first container, such as a G-REX 100MCS container, in a small-scale culture for a period of about 4 days, and then (b) transferring and distributing the T cells from the small-scale culture to two, three, or four second containers that are larger in size than the first container (e.g., a G-REX 500MCS container). In each second container, a portion of the T cells from the small-scale culture transferred to such a second container is cultured in a larger-scale culture for a period of about 5 days, and is divided into multiple steps to achieve scale-out and scale-up of the culture.

[0378]

[0432] In some embodiments, the second rapid expansion culture is performed after the activation of the T cells resulting from the first primary stimulation expansion culture begins to decrease, attenuate, decay, or calm down.

[0379]

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

[0380]

[0434] In some embodiments, after the activation of T cells brought about by the first primary stimulation expansion culture is reduced by a percentage in the range of 1% or about 1% to 100% or about 100%, a second rapid expansion culture is performed.

[0381]

[0435] In some embodiments, the activation of T cells brought about by the first primary stimulation expansion culture is reduced by a percentage in the range of 1% or about 1% to 10% or about 10%, 10% or about 10% to 20% or about 20%, 20% or about 20% to 30% or about 30%, 30% or about 30% to 40% or about 40%, 40% or about 40% to 50% or about 50%, 50% or about 50% to 60% or about 60%, 60% or about 60% to 70% or about 70%, 70% or about 70% to 80% or about 80%, 80% or about 80% to 90% or about 90%, or 90% or about 90% to 100% or about 100%, after which a second rapid expansion culture is performed.

[0382]

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

[0383]

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

[0384]

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

[0385]

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

[0386]

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

[0387]

[0441] In some embodiments, the first priming expansion culture 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.

[0388]

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

[0389]

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

[0390]

[0444] In some embodiments, the second rapid expansion culture of T cells is carried out 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.

[0391]

[0445] In some embodiments, the first initial stimulation expansion culture of T cells is carried out over a period of 1 day or about 1 day to 7 days or about 7 days, and the second rapid expansion culture of T cells is carried out over a period of 1 day or about 1 day to 11 days or about 11 days.

[0392]

[0446] In some embodiments, the first initial stimulation expansion culture of T cells is carried out over a period of at most 1 day or about 1 day, 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, 6 days or about 6 days or 7 days or about 7 days, and the second rapid expansion culture of T cells is carried out over a period of at most 1 day or about 1 day, 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, 6 days or about 6 days, 7 days or about 7 days, 8 days or about 8 days, 9 days or about 9 days, 10 days or about 10 days or 11 days or about 11 days.

[0393]

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

[0394]

[0448] In some embodiments, the first initial stimulation expansion culture of T cells is carried out over a period of 8 days, and the second rapid expansion culture of T cells is carried out over a period of 9 days.

[0395]

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

[0396]

[0450] In some embodiments, the first primary stimulation expansion culture of T cells is performed over a period of 7 days, and the second rapid expansion culture of T cells is performed over a period of 9 days.

[0397]

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

[0398]

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

[0399]

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

[0400]

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

[0401]

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

[0402]

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

[0403]

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

[0404]

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

[0405]

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

[0406]

[0460] An exemplary TIL process known as Process 3 (also referred to herein as GEN3) that includes some of these features is shown in FIG. 1 (in particular, for example, FIGS. 1B and / or 1C), and some of the advantages of this embodiment of the present invention over Process 2A are shown in FIGS. 1, 2, 30, and 31 (in particular, for example, FIGS. 1B and / or 1C). Two embodiments of Process 3 are shown in FIGS. 1 and 30 (in particular, for example, FIGS. 1B and / or 1C). Process 2A or Gen2 is also described in U.S. Patent Application Publication No. 2018 / 0280436A1, which is hereby incorporated by reference in its entirety.

[0407]

[0461] Generally as discussed and outlined herein, TILs are harvested from patient samples and are expanded in number prior to transplantation into the patient by manipulation using a TIL expansion culture process described herein and referred to as Gen3. In some embodiments, the TILs can optionally be genetically engineered as discussed below. In some embodiments, the TILs can be cryopreserved before or after expansion culture. After thawing, the TILs can also be restimulated to enhance their metabolism prior to injection into the patient.

[0408]

[0462] In some embodiments, as discussed in detail below along with the examples and figures, the first initial stimulation expansion culture (including the process referred to herein as pre-rapid expansion culture (pre-REP) and the process shown as step B in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is shortened to 1 to 8 days, and the second rapid expansion culture (including the process referred to herein as rapid expansion culture protocol (REP) and the process shown as step D in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is shortened to 1 to 9 days. In some embodiments, as discussed in detail below along with the examples and figures, the first initial stimulation expansion culture (including the process referred to herein as pre-rapid expansion culture (pre-REP) and the process shown as step B in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is shortened to 1 to 8 days, and the second rapid expansion culture (including the process referred to herein as rapid expansion culture protocol (REP) and the process shown as step D in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is shortened to 1 to 8 days. In some embodiments, as discussed in detail below along with the examples and figures, the first initial stimulation expansion culture (including the process referred to herein as pre-rapid expansion culture (pre-REP) and the process shown as step B in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is shortened to 1 to 7 days, and the second rapid expansion culture (including the process referred to herein as rapid expansion culture protocol (REP) and the process shown as step D in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is shortened to 1 to 9 days. In some embodiments, as discussed in detail below along with the examples and figures, the first initial stimulation expansion culture (including the process referred to herein as pre-rapid expansion culture (pre-REP) and the process shown as step B in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is 1 to 7 days, and the second rapid expansion culture (including the process referred to herein as rapid expansion culture protocol (REP) and the process shown as step D in FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) is 1 to 10 days.In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is shortened to 8 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 7 - 9 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 8 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 8 - 9 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is shortened to 7 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 7 - 8 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is shortened to 8 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 8 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 8 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 9 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 8 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 10 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 7 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) is 7 - 10 days.In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is 7 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is 8 - 10 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is 7 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is 9 - 10 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 7 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is 7 - 9 days. In some embodiments, as detailed in the following along with examples and figures, the first initial stimulation expansion culture (including the process referred to herein as pre-rapid expansion culture (pre-REP) and the process shown as step B in FIG. 1 (particularly, e.g., FIG. 1B and / or FIG. 1C)) is shortened to 1 - 7 days, and the second rapid expansion culture (including the process referred to herein as rapid expansion protocol (REP) and the process shown as step D in FIG. 85 (particularly, e.g., FIG. 85B)) is shortened to 1 - 9 days. In some embodiments, the first initial stimulation expansion culture (e.g., the expansion culture described as step B in FIG. 85 (particularly, e.g., FIG. 85B)) is shortened to 7 days, and the second rapid expansion culture (e.g., the expansion culture described in step D of FIG. 85 (particularly, e.g., FIG. 85B)) is 7 - 9 days. In some embodiments, as detailed in the following along with examples and figures, the combination of the first initial stimulation expansion culture and the second rapid expansion culture (e.g., the expansion cultures described as step B and step D in FIG. 85 (particularly, e.g., FIG. 85B)) is 14 - 16 days.In particular, certain embodiments of the present invention contemplate a first primary stimulation expansion culture step in which TILs are activated by exposure to an anti-CD3 antibody, such as OKT-3, in the presence of IL-2 or exposure to an antigen in the presence of at least IL-2 and an anti-CD3 antibody, such as OKT-3. In certain embodiments, the TILs activated in the first primary stimulation expansion culture step described above are a first TIL population, i.e., a primary cell population.

[0409]

[0463] The names of the following "steps", such as A, B, C, etc., refer to non-limiting examples of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C) and refer to specific non-limiting embodiments described herein. The order of steps in the following and in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C) is illustrative, and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps, are contemplated by the methods disclosed in the present application and herein.

[0410] A. Step A: Obtain a tumor sample from a patient

[0464] Generally, TILs are first obtained from circulating lymphocytes, such as a patient tumor sample or peripheral blood lymphocytes (including peripheral blood lymphocytes having TIL-like characteristics) ("primary TILs"), then expanded in culture to a larger population for further manipulation as described herein, optionally cryopreserved, and optionally phenotyped and metabolic parameters are determined as indicators of TIL health.

[0411]

[0465] Patient tumor samples can generally be obtained by means for obtaining, using methods known in the art, surgical resection, needle biopsy, or other samples containing a mixture of tumor and TIL cells. Generally, 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 of 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 (e.g., including 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 TILs are obtained from melanoma tumors, as reported to have particularly high levels of TILs.

[0412]

[0466] After obtaining, the tumor samples are generally fragmented into pieces of 1 to about 8 mm using sharp dissection 3 and about 2 to 3 mm 3is particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase), followed by mechanical dissociation (e.g., using a tissue dissociator). The tumor digest can be made by placing the tumor in the enzyme medium, mechanically dissociating the tumor for approximately 1 minute, then incubating at 37° C. under 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only very small tissue pieces remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL, a branched hydrophilic polysaccharide, can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference, may be used. Any of the aforementioned methods may be used in any of the embodiments described herein for a method of expanding TILs or a method of treating cancer.

[0413]

[0467] As described above, in some embodiments, TILs are obtained from solid tumors. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and is subjected to enzymatic digestion as an entire tumor. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1 - 2 hours. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1 - 2 hours at 37°C and 5% CO2. In some embodiments, the tumor is digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1 - 2 hours at 37°C and 5% CO2 while rotating. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C and 5% CO2 with constant rotation. In some embodiments, the entire tumor is combined with the enzyme to form a tumor digestion reaction mixture.

[0414]

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

[0415]

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

[0416]

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

[0417]

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

[0418]

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

[0419]

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

[0420]

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

[0421]

[0475] In some embodiments, fragmentation includes physical fragmentation, such as detachment and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is detachment. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can first be cultured from enzymatically digested tumors and tumor fragments obtained from a patient. In one embodiment, TILs can first be cultured from enzymatically digested tumors and tumor fragments obtained from a patient.

[0422]

[0476] In some embodiments, when the tumor is a solid tumor, for example, after obtaining a tumor sample in step A (provided in FIG. 1 (especially for example FIGS. 1B and / or 1C)), the tumor undergoes physical fragmentation. In some embodiments, the fragmentation is performed before cryopreservation. In some embodiments, the fragmentation is performed after cryopreservation. In some embodiments, the fragmentation is performed without any cryopreservation after obtaining the tumor. In some embodiments, the fragmentation step is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first primary stimulation expansion culture. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first primary stimulation expansion culture. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first primary stimulation expansion culture. In some embodiments, the plurality of fragments includes about 4 to about 50 fragments, and each fragment has a volume of about 27 mm 3 In some embodiments, the plurality of fragments includes about 30 to about 60 fragments having a total volume of about 1300 mm 3 to about 1500 mm 3 . In some embodiments, the plurality of fragments includes about 50 fragments having a total volume of about 1350 mm 3 . In some embodiments, the plurality of fragments includes about 50 fragments having a total mass of about 1 g to about 1.5 g. In some embodiments, the plurality of fragments includes about 4 fragments.

[0423]

[0477] In some embodiments, TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 1 mm 3 to 10 mm 3 . In some embodiments, the tumor fragments are about 1 mm 3 to 8 mm 3 . In some embodiments, the tumor fragments are about 1 mm 3 . In some embodiments, the tumor fragments are about 2 mm 3It is. In some embodiments, the tumor fragment is about 3 mm 3 It is. In some embodiments, the tumor fragment is about 4 mm 3 It is. In some embodiments, the tumor fragment is about 5 mm 3 It is. In some embodiments, the tumor fragment is about 6 mm 3 It is. In some embodiments, the tumor fragment is about 7 mm 3 It is. In some embodiments, the tumor fragment is about 8 mm 3 It is. In some embodiments, the tumor fragment is about 9 mm 3 It is. In some embodiments, the tumor fragment is about 10 mm 3 It is. In some embodiments, the tumor fragment is 1 to 4 mm × 1 to 4 mm × 1 to 4 mm. In some embodiments, the tumor fragment is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor fragment is 2 mm × 2 mm × 2 mm. In some embodiments, the tumor fragment is 3 mm × 3 mm × 3 mm. In some embodiments, the tumor fragment is 4 mm × 4 mm × 4 mm.

[0424]

[0478] In some embodiments, the tumor is fragmented to minimize the amount of bleeding, necrosis, and / or adipose tissue on each fragment. In some embodiments, the tumor is fragmented to minimize the amount of bleeding tissue on each fragment. In some embodiments, the tumor is fragmented to minimize the amount of necrotic tissue on each fragment. In some embodiments, the tumor is fragmented to minimize the amount of adipose tissue on each fragment. In certain embodiments, the step of fragmenting the tumor is an in vitro or ex vivo method.

[0425]

[0479] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without performing a sawing motion with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, the tumor digest is created by incubation in an enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor can be mechanically separated for about 1 minute. The solution can then be incubated at 37° C. under 5% CO2 for 30 minutes, and then it can be mechanically disrupted again for about 1 minute. After incubating again at 37° C. under 5% CO2 for 30 minutes, the tumor can be mechanically disrupted a third time for about 1 minute. In some embodiments, if large tissue pieces were present after the third mechanical disruption, one or two additional mechanical separations with or without an additional 30-minute incubation at 37° C. under 5% CO2 were applied to the sample. In some embodiments, if the cell suspension contained a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove such cells.

[0426]

[0480] In some embodiments, the cell suspension prior to the first primary stimulation expansion culture step is referred to as a “primary cell population” or a “freshly obtained” or “freshly isolated” cell population.

[0427]

[0481] In some embodiments, the cells are optionally frozen and cryopreserved after sample separation (e.g., after obtaining a tumor sample and / or after obtaining a cell suspension from a tumor sample) prior to proceeding to the expansion culture described in step B, as described in more detail below and illustrated in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C).

[0428] 1. Method for expanding peripheral blood lymphocytes (PBL) from peripheral blood

[0482] PBL Method 1. In one embodiment of the present invention, PBL is expanded in culture using the processes described herein. In one embodiment of the present invention, the method includes obtaining a PBMC sample from whole blood. In one embodiment, the method includes enriching T cells by separating pure T cells from PBMC using negative selection of the non-CD19+ fraction. In one embodiment, the method includes enriching T cells by separating pure T cells from PBMC using magnetic bead-based negative selection of the non-CD19+ fraction.

[0429]

[0483] In one embodiment of the present invention, PBL Method 1 is performed as follows: On day 0, a cryopreserved PBMC sample is thawed and the PBMC are counted. T cells are separated using a Human Pan T cell isolation kit and an LS column (Miltenyi Biotec).

[0430]

[0484] PBL Method 2. In one embodiment of the present invention, PBL is expanded in culture using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells from PBMC are enriched by incubating the PBMC at 37°C for at least 3 hours and then separating the non-adherent cells.

[0431]

[0485] In one embodiment of the present invention, PBL Method 2 is performed as follows: On day 0, a cryopreserved PBMC sample is thawed, and the PBMC cells are seeded at 6 million cells per well in a 6-well plate in CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, the non-adherent cells, which are PBL, are removed and counted.

[0432]

[0486] PBL Method 3. In one embodiment of the present invention, PBL is expanded in culture using PBL Method 3, which includes obtaining a PBMC sample from peripheral blood. B cells are separated using CD19+ selection, and T cells are selected using negative selection of the non-CD19+ fraction of the PBMC sample.

[0433]

[0487] In one embodiment of the present invention, the PBL method 3 is carried out as follows: On day 0, cryopreserved PBMCs derived from peripheral blood are thawed and counted. CD19+ B cells are sorted using the CD19 Multisort Kit, Human (Miltenyi Biotec). Among the non-CD19+ cell fraction, T cells are purified using the Human Pan T cell isolation kit and LS column (Miltenyi Biotec).

[0434]

[0488] In one embodiment, PBMCs are separated from a whole blood sample. In one embodiment, the PBMC sample is used as a starting material for expanding the culture of PBL. In one embodiment, the sample is cryopreserved prior to the expansion culture process. In another embodiment, a fresh sample is used as a starting material for expanding the culture of PBL. In one embodiment of the present invention, T cells are separated from PBMCs using methods known in the art. In one embodiment, T cells are separated using the Human Pan T cell isolation kit and LS column. In one embodiment of the present invention, T cells are separated from PBMCs using an antibody selection method known in the art, such as CD19 negative selection.

[0435]

[0489] In one embodiment of the present invention, the PBMC sample is incubated for a certain period of time at a desired temperature effective for identifying non-adherent cells. In one embodiment of the present invention, the incubation time is about 3 hours. In one embodiment of the present invention, the temperature is about 37°C. The non-adherent cells are then expanded using the above process.

[0436]

[0490] In some embodiments, the PBMC sample is from a subject or patient optionally pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or 1 year or more. In another embodiment, the PBMC is from a patient currently receiving an ITK inhibitor regimen such as ibrutinib.

[0437]

[0491] In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and resistant to treatment with a kinase inhibitor or an ITK inhibitor such as ibrutinib.

[0438]

[0492] In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but no longer receiving treatment with a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but not having received treatment with a kinase inhibitor or an ITK inhibitor for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or at least 1 year or more. In another embodiment, the PBMC is from a patient with prior exposure to an ITK inhibitor but not treated for at least 3 months, at least 6 months, at least 9 months or at least 1 year.

[0439]

[0493] In an embodiment of the present invention, on day 0, cells are selected for CD19+ and appropriately sorted. In one embodiment of the present invention, the selection is performed using antibody-conjugated beads. In one embodiment of the present invention, pure T cells are isolated from PBMC on day 0.

[0440]

[0494] In one embodiment of the present invention, for patients not pretreated with ibrutinib or other ITK inhibitors, 10 - 15 ml of buffy coat yields approximately 5×10 9 cells of PBMC, which thus yields approximately 5.5×10 7 cells of PBL.

[0441]

[0495] In one embodiment of the present invention, for patients pretreated with ibrutinib or other ITK inhibitors, the expansion culture process yields approximately 20×10 9 cells of PBL. In one embodiment of the present invention, from 40.3×10 6 cells of PBMC, approximately 4.7×10 5 cells of PBL are obtained.

[0442]

[0496] In any of the foregoing embodiments, PBMC can be obtained from a whole blood sample by apheresis, from a buffy coat, or from any other method known in the art for obtaining PBMC.

[0443] 2. Method for expanding bone marrow-infiltrating lymphocytes (MIL) from bone marrow-derived PBMC

[0497] MIL method 3. In one embodiment of the present invention, the method includes obtaining PBMC from bone marrow. On day 0, PBMC are selected for CD3+ / CD33+ / CD20+ / CD14+, sorted, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is returned to the selected cell fraction.

[0444]

[0498] In one embodiment of the present invention, the MIL method 3 is implemented as follows: On day 0, a sample of cryopreserved PBMCs is thawed and the PBMCs are counted. The cells are stained with CD3, CD33, CD20, and CD14 antibodies and sorted using S3e cell sorting (Bio-Rad). The cells are sorted into two fractions: an immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and an AML blast cell fraction (non-CD3+CD33+CD20+CD14+).

[0445]

[0499] In one embodiment of the present invention, PBMCs are obtained from bone marrow. In one embodiment, PBMCs are obtained from bone marrow through apheresis, aspiration, needle biopsy, or other similar means known in the art. In one embodiment, PBMCs are fresh. In another embodiment, PBMCs are cryopreserved.

[0446]

[0500] In one embodiment of the present invention, MIL is expanded from 10 - 50 ml of bone marrow aspirate. In one embodiment of the present invention, 10 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 20 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 30 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 40 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 50 ml of bone marrow aspirate is obtained from a patient.

[0447]

[0501] In one embodiment of the present invention, the number of PBMCs obtained from about 10 - 50 ml of bone marrow aspirate is about 5×10 7 ~ about 10×10 7 PBMCs. In another embodiment, the number of PBMCs obtained is about 7×10 7 PBMCs.

[0448]

[0502] In one embodiment of the present invention, from about 5×10 7 ~ about 10×10 7 PBMCs, about 0.5×10 6 ~ about 1.5×10 6Individuals of MIL are obtained. In one embodiment of the present invention, about 1×10 6 Individuals of MIL are obtained.

[0449]

[0503] In one embodiment of the present invention, approximately 1.4×10 6 individuals of MIL are obtained from 12×10 5 individuals of PBMC derived from bone marrow aspirate.

[0450]

[0504] In any of the above-described embodiments, PBMC can be obtained from a whole blood sample, from bone marrow, by apheresis, from a buffy coat, or by any other method known in the art for obtaining PBMC.

[0451] B. Step B: First primary stimulation expansion culture

[0505] In some embodiments, the method provides younger TILs, which may provide additional therapeutic advantages over more mature TILs (i.e., TILs that have undergone more replications prior to administration to the subject / patient). The characteristics of welded TILs are described in the literature. For example, Donia, et al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley at al., Clin Cancer Res, 16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother 32:415-423 (2009); Robbins, et al., J Immunol 2004; 173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, et al., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother, 31:742-751 (2008), all of which are hereby incorporated by reference in their entirety.

[0452]

[0506] After tumor fragments and / or dissection or digestion of tumor fragments, for example, as described in step A of FIG. 1 (particularly FIGS. 1B and / or 1C), the obtained cells are cultured in serum-containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting feeder cells) under conditions favorable for the growth of TILs over tumor and other cells. In some embodiments, IL-2, OKT-3, and feeder cells are added at the start of culture (e.g., on day 0) together with the tumor digest and / or tumor fragments. In some embodiments, the tumor digest and / or tumor fragments are incubated in a container with up to 60 fragments per container and 6000 IU / mL of IL-2. In some embodiments, this primary cell population is cultured for several days, generally over a period of 1 - 8 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells are obtained. In some embodiments, this primary cell population is cultured for several days, generally over a period of 1 - 7 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells are obtained. In some embodiments, the first primary stimulation expansion culture occurs over a period of 1 - 8 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of 1 - 7 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of about 5 - 8 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of 5 - 7 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of about 6 - 8 days, thereby obtaining a bulk TIL population, generally about 1×10 8Individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of about 6 - 7 days, thereby resulting in a bulk TIL population, generally about 1×10 8 Individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of about 7 - 8 days, thereby resulting in a bulk TIL population, generally about 1×10 8 Individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of about 7 days, thereby resulting in a bulk TIL population, generally about 1×10 8 Individual bulk TIL cells are obtained. In some embodiments, this first primary stimulation expansion culture occurs over a period of about 8 days, thereby resulting in a bulk TIL population, generally about 1×10 8 Individual bulk TIL cells are obtained.

[0453]

[0507] In preferred embodiments, as described below and herein, a first primary stimulation expansion culture step (e.g., referred to as pre-REP or primary stimulation REP, which may include a process containing feeder cells from day 0 and / or at the start of culture, such as that described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)), and then, as described below under step D and herein, a second rapid expansion culture (step D, a process including a rapid expansion culture protocol (REP) step), and then, as described below and herein, an optional cryopreservation and then a second step D (a process including a restimulation REP step) can be used to perform the expansion culture of TIL. The TIL obtained by this process can optionally be characterized by phenotypic features and metabolic parameters as described herein. In some embodiments, the tumor fragments are about 1 mm 3 ~10 mm 3 in size.

[0454]

[0508] In some embodiments, the first expansion culture medium is referred to as "CM", which is the abbreviation of the culture medium. In some embodiments, the CM in step B consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg / mL gentamicin.

[0455]

[0509] In some embodiments, there are 240 or fewer tumor fragments. In some embodiments, there are 240 or fewer tumor fragments placed in 4 or fewer containers. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, 60 or fewer tumor fragments are placed in one container. In some embodiments, each container contains 500 mL or less of the medium per container. In some embodiments, the medium contains IL-2. In some embodiments, the medium contains 6000 IU / mL of IL-2. In some embodiments, the medium contains antigen-presenting feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, the medium contains 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains OKT-3. In some embodiments, the medium contains 30 ng / mL of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng of OKT-3, and 2.5×10 8 antigen-presenting feeder cells. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen-presenting feeder cells per container.

[0456]

[0510] After preparation of the tumor fragments, the resulting cells (i.e., the fragments which are the primary cell population) are cultured in a medium containing IL-2, antigen-presenting feeder cells, and OKT-3 under conditions that are favorable for the growth of TIL over that of the tumor and other cells, enabling the acceleration of TIL primary stimulation and growth from the start of culture on day 0. In some embodiments, the tumor digest and / or tumor fragments are incubated with 6000 IU / mL of IL-2 along with antigen-presenting feeder cells and OKT-3. This primary cell population is cultured over a period of several days, generally 1 - 8 days, thereby yielding a bulk TIL population, generally about 1×10 8 bulk TIL cells are obtained. In some embodiments, the growth medium during the first primary stimulation expansion culture contains IL-2 or a variant thereof along with antigen-presenting feeder cells and OKT-3. In some embodiments, this primary cell population is cultured over a period of several days, generally 1 - 7 days, thereby yielding a bulk TIL population, generally about 1×10 8 bulk TIL cells are obtained. In some embodiments, the growth medium during the first primary stimulation expansion culture contains IL-2 or a variant thereof along with antigen-presenting feeder cells and OKT-3. In some embodiments, IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution has a specific activity of 20 - 30×10 6 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 20×10 6 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 25×10 6 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 30×10 6 IU / mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 4 - 8×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 5 - 7×10 6 IU / mg. In some embodiments, the IL-2 stock solution has a final concentration of IL-2 of 6×10 6It has a final concentration of IL-2 of IU / mg. In some embodiments, the IL-2 stock solution is prepared as described in Example C. In some embodiments, the first primary stimulation expansion culture medium contains about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6,000 IU / mL of IL-2 or about 5,000 IU / mL of IL-2. In some embodiments, the first primary stimulation expansion culture medium contains from about 9,000 IU / mL to about 5,000 IU / mL of IL-2. In some embodiments, the first primary stimulation expansion culture medium contains from about 8,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first primary stimulation expansion culture medium contains from about 7,000 IU / mL to about 6,000 IU / mL of IL-2. In some embodiments, the first primary stimulation expansion culture medium contains about 6,000 IU / mL of IL-2. In one embodiment, the cell culture medium further contains IL-2. In some embodiments, the first primary stimulation expansion culture cell medium contains about 3,000 IU / mL of IL-2. In one embodiment, the first primary stimulation expansion culture cell culture medium further contains IL-2. In a preferred embodiment, the first primary stimulation expansion culture cell culture medium contains about 3,000 IU / mL of IL-2. In one embodiment, the first primary stimulation expansion culture cell culture medium contains about 1,000 IU / mL, about 1,500 IU / mL, about 2,000 IU / mL, about 2,500 IU / mL, about 3,000 IU / mL, about 3,500 IU / mL, about 4,000 IU / mL, about 4,500 IU / mL, about 5,000 IU / mL, about 5,500 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL or about 8,000 IU / mL of IL-2. In one embodiment, the first primary stimulation expansion culture cell culture medium contains from 1,000 to 2,000 IU / mL, from 2,000 to 3,000 IU / mL, from 3,000 to 4,000 IU / mL, from 4,000 to 5,000 IU / mL, from 5,000 to 6,000 IU / mL, from 6,000 to 7,000 IU / mL, from 7,000 to 8,000 IU / mL or about 8,000 IU / mL of IL-2.

[0457]

[0511] In some embodiments, the first initial stimulation expansion culture medium contains IL-15 at about 500 IU / mL, about 400 IU / mL, about 300 IU / mL, about 200 IU / mL, about 180 IU / mL, about 160 IU / mL, about 140 IU / mL, about 120 IU / mL, or about 100 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-15 from about 500 IU / mL to about 100 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-15 from about 400 IU / mL to about 100 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-15 from about 300 IU / mL to about 100 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-15 at about 200 IU / mL. In some embodiments, the first initial stimulation expansion cell culture medium contains IL-15 at about 180 IU / mL. In one embodiment, the first initial stimulation expansion cell culture medium further contains IL-15. In a preferred embodiment, the first initial stimulation expansion cell culture medium contains IL-15 at about 180 IU / mL.

[0458]

[0512] In some embodiments, the first initial stimulation expansion culture medium contains IL-21 at about 20 IU / mL, about 15 IU / mL, about 12 IU / mL, about 10 IU / mL, about 5 IU / mL, about 4 IU / mL, about 3 IU / mL, about 2 IU / mL, about 1 IU / mL or about 0.5 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-21 from about 20 IU / mL to about 0.5 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-21 from about 15 IU / mL to about 0.5 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-21 from about 12 IU / mL to about 0.5 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-21 from about 10 IU / mL to about 0.5 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-21 from about 5 IU / mL to about 1 IU / mL. In some embodiments, the first initial stimulation expansion culture medium contains IL-21 at about 2 IU / mL. In some embodiments, the first initial stimulation expansion cell culture medium contains IL-21 at about 1 IU / mL. In some embodiments, the first initial stimulation expansion cell culture medium contains IL-21 at about 0.5 IU / mL. In one embodiment, the cell culture medium further contains IL-21. In a preferred embodiment, the first initial stimulation expansion cell culture medium contains IL-21 at about 1 IU / mL.

[0459]

[0513] In one embodiment, the first primary stimulation expansion culture cell culture medium contains OKT-3 antibody. In some embodiments, the first primary stimulation expansion culture cell culture medium contains about 30 ng / mL of OKT-3 antibody. In one embodiment, the first primary stimulation expansion culture cell culture medium contains OKT-3 antibody at 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. In one embodiment, the cell culture medium contains OKT-3 antibody at 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL and 50 ng / mL to 100 ng / mL. In one embodiment, the cell culture medium contains OKT-3 antibody at 15 ng / mL to 30 ng / mL. In one embodiment, the cell culture medium contains about 30 ng / mL of OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0460]

Table 4

[0461]

[0514] In some embodiments, the first primary stimulation expansion culture cell culture medium contains 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, utomilumab, EU-101, fusion proteins and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL in the cell culture medium.

[0462]

[0515] In some embodiments, in addition to one or more TNFRSFs, the first primary stimulation expansion culture cell culture medium further contains 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 the one or more TNFRSF agonists include a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSFs, the first primary stimulation expansion culture cell culture medium further contains IL-2 at an initial concentration of about 6000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists include a 4-1BB agonist.

[0463]

[0516] In some embodiments, the first primary stimulation expansion culture medium is referred to as "CM", which is an abbreviation of the culture medium. In some embodiments, this is referred to as CM1 (culture medium 1). In some embodiments, CM consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg / μL gentamicin. In some embodiments, CM is CM1 as described in the examples; see Example A. In some embodiments, the first primary stimulation expansion culture is performed in an initial cell culture medium or a first cell culture medium. In some embodiments, the first primary stimulation expansion culture medium or the initial cell culture medium or the first cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells (also referred to as feeder cells).

[0464]

[0517] In some embodiments, the culture medium used in the expansion culture process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium contains a basal cell medium and a serum supplement and / or a serum substitute. In some embodiments, the serum-free or defined medium is used to prevent and / or reduce experimental variations resulting from variations between lots of partially serum-containing media.

[0465]

[0518] In some embodiments, the serum-free or defined medium contains a basal cell medium and a serum supplement and / or a serum substitute. In some embodiments, the basal cell medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, Iscove's Modified Dulbecco Medium.

[0466]

[0519] In some embodiments, the serum supplement or serum replacement includes, but is not limited to, one or more CTS(™) OpTmizer(™) T Cell Expansion Serum Supplements, CTS(™) Immune Cell Serum Replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium includes albumin, glycine, L - histidine, L - isoleucine, L - methionine, L - phenylalanine, L - proline, L - hydroxyproline, L - serine, L - threonine, L - tryptophan, L - tyrosine, L - valine, thiamine, reduced glutathione, L - ascorbic acid - 2 - phosphate, iron - saturated transferrin, insulin, and the trace element moiety Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3 ”, Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ and Zr 4+ and one or more components selected from the group consisting of compounds containing the same. In some embodiments, the defined medium further includes L - glutamine, sodium bicarbonate, and / or 2 - mercaptoethanol.

[0467]

[0520] In some embodiments, the CTS(Trademark) OpTmizer(Trademark) T-cell immune cell serum replacement is used with conventional growth media including, but not limited to, CTS(Trademark) OpTmizer(Trademark) T-cell growth basal medium, CTS(Trademark) OpTmizer(Trademark) T-cell growth SFM, CTS(Trademark) AIM-V medium, CST(Trademark) AIM-V SFM, LymphoONE(Trademark) T-cell growth xenofree medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI growth medium, Iscove's Modified Dulbecco Medium.

[0468]

[0521] In some embodiments, the total serum replacement concentration (volume %) in the serum-free or defined medium is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the total volume of the serum-free or defined medium. 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.

[0469]

[0522] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, and is mixed prior to use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0470]

[0523] In some embodiments, the limited medium is CTS(™) OpTmizer(™) T Cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS(™) OpTmizer(™) is useful in the present invention. CTS(™) OpTmizer(™) T Cell Expansion SFM is a combination of 1 L of CTS(™) OpTmizer(™) T Cell Expansion Basal Medium and 26 mL of CTS(™) OpTmizer(™) T Cell Expansion Supplement, which is mixed before use. In some embodiments, CTS(™) OpTmizer(™) T Cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, CTS(™) OpTmizer(™) T Cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine. In some embodiments, CTS(™) OpTmizer(™) T Cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and further contains IL-2 at about 1000 IU / mL to about 8000 IU / mL. In some embodiments, CTS(™) OpTmizer(™) T Cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and further contains about 3000 IU / mL of IL-2. In some embodiments, CTS(™) OpTmizer(™) T Cell Expansion SFM is supplemented with approximately 3% CTS(™) Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and further contains about 6000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains from about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0471]

[0524] In some embodiments, the serum-free medium or the defined medium is supplemented with glutamine (i.e., GlutaMAX™) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or the defined medium is supplemented with glutamine (i.e., GlutaMAX™) at a concentration of 2 mM.

[0472]

[0525] In some embodiments, the serum-free medium or the 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 the defined medium is supplemented with 2-mercaptoethanol at a concentration of 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0473]

[0526] In some embodiments, the defined medium described in International Publication No. WO 1998 / 030679, which is incorporated herein by reference, is useful in the present invention. That publication describes a serum-free eukaryotic cell culture medium. Serum-free eukaryotic cell culture media include a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. Serum-free eukaryotic cell culture medium supplements include one or more albumin 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 insulin or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics, or are obtained by combining them. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and / or β-mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag + 、Al 3+ 、Ba 2+ 、Cd 2+ 、Co 2+ 、Cr 3 ”、Ge 4+ 、Se 4+ 、Br、T、Mn 2+ 、P、Si 4+ 、V 5+ 、Mo 6+ 、Ni 2+ 、Rb + 、Sn2+ and Zr 4+ and one or more components selected from the group consisting of compounds containing 4+ . In some embodiments, the basal cell medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco Medium.

[0474]

[0527] In some embodiments, the concentration of glycine in the defined medium ranges from about 5 to 200 mg / L, the concentration of L-histidine is about 5 to 250 mg / L, the concentration of L-isoleucine is about 5 to 300 mg / L, the concentration of L-methionine is about 5 to 200 mg / L, the concentration of L-phenylalanine is about 5 to 400 mg / L, the concentration of L-proline is about 1 to 1000 mg / L, the concentration of L-hydroxyproline is about 1 to 45 mg / L, the concentration of L-serine is about 1 to 250 mg / L, the concentration of L-threonine is about 10 to 500 mg / L, the concentration of L-tryptophan is about 2 to 110 mg / L, the concentration of L-tyrosine is about 3 to 175 mg / L, the concentration of L-valine is about 5 to 500 mg / L, the concentration of thiamine is about 1 to 20 mg / L, the concentration of reduced glutathione is about 1 to 20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg / L, the concentration of iron-saturated transferrin is about 1 to 50 mg / L, the concentration of insulin is about 1 to 100 mg / L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg / L, and the concentration of albumin (such as AlbuMAX® I) is about 5000 to 50,000 mg / L.

[0475]

[0528] In some embodiments, the non-trace element sub-components in the defined medium are present in the concentration ranges listed in the column under the heading "Concentration Range in 1X Medium" of Table A1 below. In other embodiments, the non-trace element sub-components in the defined medium are present at the final concentrations listed in the column under the heading "Preferred Embodiment of 1X Medium" of Table A1 below. In other embodiments, the defined medium is a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-trace element sub-components of the types and concentrations listed in the column under the heading "Preferred Embodiment of Supplement" of Table A1 below.

[0476]

Table 5

[0477]

[0529] In some embodiments, the volume osmolality of the defined medium is about 260 - 350 mOsmol. In some embodiments, the volume osmolality is about 280 - 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L or about 2.2 g / L of sodium bicarbonate. The defined medium can be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0478]

[0530] 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) is useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

[0479]

[0531] In one embodiment, the cell culture medium in the first and / or second gas permeable container is not filtered. The use of non-filtered cell culture medium can simplify the procedures necessary to expand the cell number by culture. In one embodiment, the cell culture medium in the first and / or second gas permeable container lacks beta-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

[0480]

[0532] In some embodiments, as discussed in the examples and figures, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 1 to 7 days. In some embodiments, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 2 to 8 days. In some embodiments, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 3 to 8 days. In some embodiments, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 4 to 8 days. In some embodiments, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 5 to 7 days. In some embodiments, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 2 to 8 days. In some embodiments, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 2 to 7 days. In some embodiments, the first primary stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or primary stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C)) process is from 3 to 8 days.In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 3 to 7 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 4 to 8 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 4 to 7 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 5 to 8 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 5 to 7 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 6 to 8 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those described in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 6 to 7 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, including processes such as those provided in step B of FIG. 1 (particularly, for example, FIG. 1B and / or FIG. 1C)) process is 7 to 8 days.In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, such as the process provided in step B of FIG. 1 (particularly for example FIGS. 1B and / or 1C)) process is 8 days. In some embodiments, the first initial stimulation expansion culture (which may include, for example, what may also be referred to as pre-REP or initial stimulation REP, such as the process provided in step B of FIG. 1 (particularly for example FIGS. 1B and / or 1C)) process is 7 days.

[0481]

[0533] In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 1 to 7 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 2 to 7 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 3 to 7 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 3 to 8 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 3 to 7 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 4 to 8 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 4 to 7 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 5 to 8 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 5 to 7 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 6 to 8 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 6 to 7 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 7 to 8 days from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated.In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 8 days starting from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated. In some embodiments, the first primary-stimulated TIL expansion culture can be continued for 7 days starting from when fragmentation occurs and / or when the first primary-stimulated expansion culture step is initiated.

[0482]

[0534] In some embodiments, the first primary-stimulated expansion culture of TIL can be continued 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 culture can be continued for 1 to 7 days. In some embodiments, the first TIL expansion culture can be continued for 2 to 8 days. In some embodiments, the first TIL expansion culture can be continued for 2 to 7 days. In some embodiments, the first TIL expansion culture can be continued for 3 to 8 days. In some embodiments, the first TIL expansion culture can be continued for 3 to 7 days. In some embodiments, the first TIL expansion culture can be continued for 4 to 8 days. In some embodiments, the first TIL expansion culture can be continued for 4 to 7 days. In some embodiments, the first TIL expansion culture can be continued for 5 to 8 days. In some embodiments, the first TIL expansion culture can be continued for 5 to 7 days. In some embodiments, the first TIL expansion culture can be continued for 6 to 8 days. In some embodiments, the first TIL expansion culture can be continued for 6 to 7 days. In some embodiments, the first TIL expansion culture can be continued for 8 days. In some embodiments, the first TIL expansion culture can be continued for 7 days.

[0483]

[0535] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination during the first priming expansion culture. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21 and any combination thereof can be included during the first priming expansion culture, including, for example, during the step B process as shown in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C) and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the first priming expansion culture. In some embodiments, IL-2, IL-15, and IL-21 and any combination thereof can be included, for example, during the step B process as shown in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C) and as described herein.

[0484]

[0536] In some embodiments, the first priming expansion culture, for example, step B as shown in FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), is performed in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL expansion culture. In some embodiments, a bioreactor is used. In some embodiments, the bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

[0485] 1. Feeder cells and antigen-presenting cells

[0537] In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any time point between days 4 and 8. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any time point between days 4 and 7. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any time point between days 5 and 8. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any time point between days 5 and 7.In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly FIGS. 1B and / or 1C) and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any point on days 6 - 8. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly FIGS. 1B and / or 1C) and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any point on days 6 - 7. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly FIGS. 1B and / or 1C) and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any point on day 7 or 8. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly FIGS. 1B and / or 1C) and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any point on day 7. In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly FIGS. 1B and / or 1C) and the expansion culture referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture, but is added during the first priming expansion culture at any point on day 8.

[0486]

[0538] In one embodiment, the first priming expansion culture procedure described herein (e.g., the expansion culture described in step B of FIG. 1 (particularly FIGS. 1B and / or 1C) and the expansion culture referred to as pre-REP or priming REP) requires feeder cells (also referred to herein as "antigen-presenting cells") at the start of the TIL expansion culture and during the first priming expansion culture. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic healthy donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In some embodiments, 2.5×10 8 feeder cells are used during the first priming expansion culture. In some embodiments, 2.5×10 8 feeder cells per vessel are used during the first priming expansion culture. In some embodiments, 2.5×10 8 feeder cells per GREX-10 are used during the first priming expansion culture. In some embodiments, 2.5×10 8 feeder cells per GREX-100 are used during the first priming expansion culture.

[0487]

[0539] Generally, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the REP procedure as described in the examples providing exemplary protocols for assessing the replicative ability of allogeneic PBMCs.

[0488]

[0540] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells placed in the culture on day 0 of the first priming expansion culture, the PBMCs are considered non-replicative and are approved for use in the TIL expansion culture procedure described herein.

[0489]

[0541] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 has not increased from the initial number of viable cells seeded into the culture on day 0 of the first primary stimulation expansion culture, the PBMCs are considered non-replicative and are approved for use in the TIL expansion culture procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 3000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0490]

[0542] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on day 7 has not increased from the initial number of viable cells seeded into the culture on day 0 of the first primary stimulation expansion culture, the PBMCs are considered non-replicative and are approved for use in the TIL expansion culture procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 5 - 60 ng / mL of OKT3 antibody and 1000 - 6000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 10 - 50 ng / mL of OKT3 antibody and 2000 - 5000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 20 - 40 ng / mL of OKT3 antibody and 2000 - 4000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 25 - 35 ng / mL of OKT3 antibody and 2500 - 3500 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 15 ng / mL of OKT3 antibody and 3000 IU / ml of IL-2. In some embodiments, the PBMCs are cultured in the presence of 15 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0491]

[0543] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or about 1:500. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is from 1:50 to 1:300. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is from 1:100 to 1:200.

[0492]

[0544] In one embodiment, the first primary stimulation expansion culture procedure described herein requires a ratio of about 2.5×10 8 feeder cells to about 100×10 6 TILs. In another embodiment, the first primary stimulation expansion culture procedure described herein requires a ratio of about 2.5×10 8 feeder cells to about 50×10 6 TILs. In yet another embodiment, the first primary stimulation expansion culture described herein requires about 2.5×10 8 feeder cells and about 25×10 6 TILs. In yet another embodiment, the first primary stimulation expansion culture described herein requires about 2.5×10 8 feeder cells. In yet another embodiment, the first primary stimulation expansion culture requires one-fourth, one-third, five-twelfths or one-half of the number of feeder cells used in the second rapid expansion culture.

[0493]

[0545] In some embodiments, the medium in the first primary stimulation expansion culture contains IL-2. In some embodiments, the medium in the first primary stimulation expansion culture contains 6000 IU / mL of IL-2. In some embodiments, the medium in the first primary stimulation expansion culture contains antigen-presenting feeder cells. In some embodiments, the medium in the first primary stimulation expansion culture contains 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium in the first primary stimulation expansion culture contains OKT-3. In some embodiments, the medium contains 30 ng of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen-presenting feeder cells. In some embodiments, the medium contains 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains 500 mL of culture medium and 15 μg of OKT-3 per 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains 500 mL of culture medium and 15 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium contains 500 mL of culture medium, 6000 IU / mL of IL-2, 30 ng / mL of OKT-3, and 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains 500 mL of culture medium, 6000 IU / mL of IL-2, 15 μg of OKT-3, and 2.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium contains 500 mL of culture medium and 15 μg of OKT-3 per 2.5×10 8 antigen-presenting feeder cells.

[0494]

[0546] In one embodiment, the first priming expansion culture procedure described herein requires an excess amount of feeder cells for TILs during the second expansion culture. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic healthy donor. 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.

[0495]

[0547] Generally, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the TIL expansion culture procedures described herein, including the exemplary procedures described in the figures and examples.

[0496]

[0548] In one embodiment, artificial antigen-presenting cells are used in the first priming expansion culture as an alternative to or in combination with PBMCs.

[0497] 2. Cytokines

[0549] The expansion culture methods described herein generally use a culture medium containing high doses of cytokines, particularly IL-2, as is known in the art.

[0498]

[0550] Alternatively, it is further possible to use a combination of cytokines for the first priming expansion culture of TILs, and for combinations of two or more of IL-2, IL-15, and IL-21, it is generally as outlined in International Publication No. WO 2015 / 189356 and International Publication No. WO 2015 / 189357, which are hereby expressly incorporated by reference in their entirety. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, and the latter find particular use in many embodiments. As described in these specifications, using a combination of cytokines is advantageous, particularly for the generation of lymphocytes, specifically T cells.

[0499]

Table 6

[0500] C. Step C: Transition from the First Primary Expansion Culture to the Second Rapid Expansion Culture

[0551] In some cases, for example, the bulk TIL population obtained from the first primary expansion culture, including the TIL population obtained from step B as shown in FIG. 1 (particularly FIGS. 1B and / or 1C), which may include an expansion culture sometimes referred to as a pre-REP, can be subjected to a second rapid expansion culture (which may include an expansion culture sometimes referred to as a rapid expansion protocol (REP)) and then cryopreserved as discussed below. Similarly, when genetically modified TILs are used for treatment, the expanded TIL population from the primary expansion culture or the expanded TIL population from the second rapid expansion culture can be subjected to genetic modification for appropriate treatment before the expansion step or after the first primary expansion culture and before the second rapid expansion culture.

[0501]

[0552] In some embodiments, TILs obtained from the first primary stimulation expansion culture (e.g., from step B as shown in FIG. 1 (particularly FIGS. 1B and / or 1C)) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first primary stimulation expansion culture (e.g., from step B as shown in FIG. 1 (particularly FIGS. 1B and / or 1C)) are not stored and proceed directly to the second rapid expansion culture. In some embodiments, TILs obtained from the first primary stimulation expansion culture are not cryopreserved after the first primary stimulation expansion culture and before the second rapid expansion culture. In some embodiments, the transfer from the first primary stimulation expansion culture to the second expansion culture occurs at about 2, 3, 4, 5, 6, 7, or 8 days from when the tumor fragment occurred and / or when the first primary stimulation expansion culture step was initiated. In some embodiments, the transfer from the first primary stimulation expansion culture to the second rapid expansion culture occurs at about 3 - 7 days from when the core or fragment was added to the cell culture medium and / or when the first primary stimulation expansion culture step was initiated. In some embodiments, the transfer from the first primary stimulation expansion culture to the second rapid expansion culture occurs at about 3 - 7 days from when fragmentation occurred and / or when the first primary stimulation expansion culture step was initiated. In some embodiments, the transfer from the first primary stimulation expansion culture to the second expansion culture occurs at about 3 - 8 days from when fragmentation occurred and / or when the first primary stimulation expansion culture step was initiated. In some embodiments, the transfer from the first primary stimulation expansion culture to the second expansion culture occurs at about 4 - 7 days from when fragmentation occurred and / or when the first primary stimulation expansion culture step was initiated. In some embodiments, the transfer from the first primary stimulation expansion culture to the second expansion culture occurs at about 4 - 8 days from when fragmentation occurred and / or when the first primary stimulation expansion culture step was initiated. In some embodiments, the transfer from the first primary stimulation expansion culture to the second expansion culture occurs at about 5 - 7 days from when fragmentation occurred and / or when the first primary stimulation expansion culture step was initiated.

[0502]

[0553] In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed at about 6 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed at about 6 to 7 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second expansion culture is performed at about 6 to 8 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second expansion culture is performed at about 7 to 8 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed at about 7 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second expansion culture is performed at about 8 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is initiated.

[0503]

[0554] In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed at 1, 2, 3, 4, 5, 6, 7, or 8 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed from 1 to 7 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed from 1 to 8 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second expansion culture is performed from 2 to 7 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second expansion culture is performed from 2 to 8 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second expansion culture is performed from 3 to 7 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second expansion culture is performed from 3 to 8 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed from 4 to 7 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed from 4 to 8 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is performed from 5 to 7 days from when fragmentation occurred and / or when the first initial stimulation expansion culture step was initiated.In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is carried out in 5 to 8 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is started. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is carried out in 6 to 7 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is started. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is carried out in 6 to 8 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is started. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is carried out in 7 to 8 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is started. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is carried out in 7 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is started. In some embodiments, the transition from the first initial stimulation expansion culture to the second rapid expansion culture is carried out in 8 days from when fragmentation occurs and / or when the first initial stimulation expansion culture step is started.

[0504]

[0555] In some embodiments, the TILs are not stored after the first initial stimulation expansion culture and before the second rapid expansion culture, and the TILs proceed directly to the second rapid expansion culture (e.g., in some embodiments, as shown in FIG. 1 (particularly for example FIGS. 1B and / or 1C), no storage is carried out during the transition from step B to step D). In some embodiments, as described herein, the transition is carried out in a closed system. In some embodiments, the TILs from the first initial stimulation expansion culture are the TILs from the second TIL population and proceed directly to the second rapid expansion culture without a transition period.

[0505]

[0556] In some embodiments, the transition from the first priming expansion culture to the second rapid expansion culture, e.g., step C according to FIG. 1 (particularly e.g., FIG. 1B), is performed in a closed - system bioreactor. In some embodiments, as described herein, a closed system is used for TIL expansion culture. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G - REX - 10 or G - REX - 100. In some embodiments, the closed - system bioreactor is a single bioreactor. In some embodiments, the transition from the first priming expansion culture to the second rapid expansion culture involves a scale - up of the container size. In some embodiments, the first priming expansion culture is performed in a container smaller than the second rapid expansion culture. In some embodiments, the first priming expansion culture is performed in a GREX - 100 and the second rapid expansion culture is performed in a GREX - 500.

[0506] D. Step D: Second rapid expansion culture

[0557] In some embodiments, the TIL cell population further increases in number after recovery and the first priming expansion culture, after the transitions referred to as steps A, B, and step C, as shown, for example, in FIG. 1 (particularly e.g., FIGS. 1B and / or 1C). This further increase is herein referred to as the second rapid expansion culture, which can include an expansion culture process generally referred to in the art as the rapid expansion culture method (rapid expansion culture protocol or REP and the process shown in step D of FIG. 1 (particularly e.g., FIGS. 1B and / or 1C)). The second rapid expansion culture can generally be achieved using a culture medium in a gas - permeable container containing several components, including feeder cells, cytokine sources, and anti - CD3 antibody. In some embodiments, 1, 2, 3, or 4 days after the start of the second rapid expansion culture (i.e., days 8, 9, 10, or 11 of the overall Gen3 process), the TILs are transferred to a larger - volume container.

[0507]

[0558] In some embodiments, the second rapid expansion culture of TIL (which may also be referred to as an expansion culture called REP; and may include the process shown in step D of FIG. 1 (particularly for example FIGS. 1B and / or 1C)) can be carried out using any TIL flask or container known to those skilled in the art. In some embodiments, the second TIL expansion culture can continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 1 day to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 1 day to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 2 days to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 2 days to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 3 days to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 3 days to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 4 days to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 4 days to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 5 days to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 5 days to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 6 days to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 6 days to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 7 days to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 7 days to about 10 days after the start of the second rapid expansion culture.In some embodiments, the second TIL expansion culture can continue for about 8 to about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 8 to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 9 to about 10 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 1 day after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 2 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 3 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 4 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 5 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 6 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 7 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 8 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 9 days after the start of the second rapid expansion culture. In some embodiments, the second TIL expansion culture can continue for about 10 days after the start of the second rapid expansion culture.

[0508]

[0559] In one embodiment, the second rapid expansion culture can be performed in a gas-permeable container using the methods of the present disclosure (e.g., including the expansion referred to as REP and the process shown in step D of FIG. 1 (particularly FIGS. 1B and / or 1C). In some embodiments, the TILs are expanded in the second rapid expansion culture in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as “antigen-presenting cells”). In some embodiments, the TILs are expanded in the second rapid expansion culture in the presence of IL-2, OKT-3, and feeder cells, where the feeder cells are added to a final concentration that is 2-fold, 2.4-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold the concentration of the feeder cells present in the first priming expansion culture. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Examples of non-specific T cell receptor stimulation include anti-CD3 antibodies such as OKT3 at about 30 ng / ml, mouse monoclonal anti-CD3 antibodies (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA), or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further stimulation of the TILs in vitro by inducing one or more antigens of cancer, including their antigenic portions, such as one or more epitopes, for example, 0.3 μM MART-1:26-35 (27L) or gpl00:209-217 (210M), which can optionally be expressed from a vector, during the second expansion culture in the presence of a T cell growth factor such as 300 IU / mL IL-2 or IL-15. Other suitable antigens can include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or their antigenic portions. TILs can also be rapidly expanded by restimulation with the same one or more antigens of cancer pulsed onto HLA-A2-expressing antigen-presenting cells.Alternatively, the TILs can be further restimulated, for example, by irradiated autologous lymphocytes or by irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second expansion culture. In some embodiments, the second expansion culture occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0509]

[0560] 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 1000 - 2000 IU / mL, 2000 - 3000 IU / mL, 3000 - 4000 IU / mL, 4000 - 5000 IU / mL, 5000 - 6000 IU / mL, 6000 - 7000 IU / mL, 7000 - 8000 IU / mL or 8000 IU / mL of IL-2.

[0510]

[0561] In one embodiment, the cell culture medium contains an OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains OKT-3 antibodies at 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. In one embodiment, the cell culture medium contains OKT-3 antibodies at 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL. In one embodiment, the cell culture medium contains OKT-3 antibodies at 30 ng / mL to 60 ng / mL. In one embodiment, the cell culture medium contains about 60 ng / mL of OKT-3. In some embodiments, the OKT-3 antibody is muromonab.

[0511]

[0562] In some embodiments, the medium in the second rapid expansion culture contains IL-2. In some embodiments, the medium contains 6000 IU / mL of IL-2. In some embodiments, the medium in the second rapid expansion culture contains antigen-presenting feeder cells. In some embodiments, the medium in the second rapid expansion culture contains 7.5×10 8 antigen-presenting feeder cells per container. In some embodiments, the medium in the second rapid expansion culture contains OKT-3. In some embodiments, the second rapid expansion culture medium contains 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium in the second rapid expansion culture contains 6000 IU / mL of IL-2, 60 ng / mL of OKT-3, and 7.5×108 contains antigen - presenting feeder cells. In some embodiments, the medium contains 500 mL of culture medium, 6000 IU / mL of IL - 2, 30 μg of OKT - 3 and 7.5×10 8 antigen - presenting feeder cells per container.

[0512]

[0563] In some embodiments, the medium in the second rapid expansion culture contains IL - 2. In some embodiments, the medium contains 6000 IU / mL of IL - 2. In some embodiments, the medium in the second rapid expansion culture contains antigen - presenting feeder cells. In some embodiments, the medium contains 5×10 8 ~7.5×10 8 antigen - presenting feeder cells per container. In some embodiments, the medium in the second rapid expansion culture contains OKT - 3. In some embodiments, the medium in the second rapid expansion culture contains 500 mL of culture medium and 30 μg of OKT - 3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium in the second rapid expansion culture contains 6000 IU / mL of IL - 2, 60 ng / mL of OKT - 3 and 5×10 8 ~7.5×10 8 antigen - presenting feeder cells per container. In some embodiments, the medium in the second rapid expansion culture contains 500 mL of culture medium, 6000 IU / mL of IL - 2, 30 μg of OKT - 3 and 5×10 8 ~7.5×10 8 antigen - presenting feeder cells per container.

[0513]

[0564] In some embodiments, the cell culture medium contains one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins and their fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL in the cell culture medium.

[0514]

[0565] In some embodiments, in addition to one or more TNFRSFs, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0515]

[0566] In some embodiments, a combination of IL-2, IL-7, IL-15 and / or IL-21 is used as the combination during the second expansion culture. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21 and any optional combinations thereof can be included during the second expansion culture, for example, as shown in FIG. 1 (especially FIGS. 1B and / or 1C) and as described herein, including during the step D process. In some embodiments, a combination of IL-2, IL-15 and IL-21 is used as the combination during the second expansion culture. In some embodiments, IL-2, IL-15 and IL-21 and any optional combinations thereof can be included during the step D process, for example, as shown in FIG. 1 (especially FIGS. 1B and / or 1C) and as described herein.

[0516]

[0567] In some embodiments, the second expansion culture can be carried out in a supplemented cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion culture is carried out in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion culture occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0517]

[0568] In some embodiments, the second expansion culture medium contains about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains from about 500 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains from about 400 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains from about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains about 200 IU / mL of IL-15. In some embodiments, the cell culture medium contains about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU / mL of IL-15.

[0518]

[0569] In some embodiments, the second expansion culture medium comprises about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21 or about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises from about 20 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises from about 15 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises from about 12 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises from about 10 IU / mL of IL-21 to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises from about 5 IU / mL of IL-21 to about 1 IU / mL of IL-21. In some embodiments, the second expansion culture medium comprises about 2 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU / mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU / mL of IL-21.

[0519]

[0570] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion culture and / or the second expansion culture is about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:75, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or about 1:500. In one embodiment, the ratio of TILs to PBMCs in the rapid expansion culture and / or the second expansion culture is from 1:50 to 1:300. In one embodiment, the ratio of TILs to PBMCs in the rapid expansion culture and / or the second expansion culture is from 1:100 to 1:200.

[0520]

[0571] In one embodiment, the REP and / or the second rapid expansion culture is performed in a flask by mixing bulk TILs with 100-fold or 200-fold excess of inactivated feeder cells in 150 mL of medium (where the concentration of the feeder cells 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, 2.1X, 2.2X, 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 first priming expansion culture), 30 mg / mL of OKT3 anti-CD3 antibody and 6000 IU / mL of IL-2. Medium replenishment is performed until the cells are transferred to another growth chamber (generally, 2 / 3 medium replenishment by aspiration of 2 / 3 of the used medium and replacement with an equal volume of fresh medium). The other growth chamber contains G-REX flasks and gas-permeable containers as will be considered in more detail below.

[0521]

[0572] In some embodiments, the second rapid expansion culture (which may include the process referred to as the REP process) is 7 - 9 days, as discussed in the examples and figures. In some embodiments, the second expansion culture is 7 days. In some embodiments, the second expansion culture is 8 days. In some embodiments, the second expansion culture is 9 days.

[0522]

[0573] In one embodiment, the second expansion culture (which may include the expansion culture referred to as REP; and as referred to in step D of FIG. 1, particularly for example FIGS. 1B and / or 1C) can be carried out in a 500 mL capacity gas - permeable flask with a 100 cm gas - permeable silicon bottom (G - Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL of IL - 2 and 30 ng / mL of anti - CD3 (OKT3), with 5×10 6 or 10×10 6 TILs can be cultured with PBMCs. The G - Rex 100 flask can be incubated at 37 °C under 5% CO2. On day 5, 250 mL of the supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 6000 IU / mL of IL - 2 and returned to and added to the original GREX - 100 flask. When continuously expanding TILs in the GREX - 100 flask, on day 10 or 11, the TILs can be transferred to a larger flask such as a GREX - 500. The cells can be harvested on day 14 of culture. The cells can be harvested on day 15 of culture. The cells can be harvested on day 16 of culture. In some embodiments, medium replacement is performed until the cells are transferred to another growth chamber. In some embodiments, 2 / 3 of the medium is replaced by aspiration of the used medium and replacement with an equal volume of fresh medium. In some embodiments, the other growth chamber includes GREX flasks and gas - permeable containers as discussed in more detail below.

[0523]

[0574] In some embodiments, the culture medium used in the expansion culture process disclosed herein is a serum-free medium or a chemically defined medium. In some embodiments, the serum-free or chemically defined medium comprises a basal cell medium and a serum supplement and / or a serum replacement. In some embodiments, the serum-free or chemically defined medium is used to prevent and / or reduce experimental variability due to variability between lots of serum-containing media.

[0524]

[0575] In some embodiments, the serum-free or chemically defined medium comprises a basal cell medium and a serum supplement and / or a serum replacement. In some embodiments, the basal cell medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xenofree Medium, Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI Growth Medium, Iscove's Modified Dulbecco Medium.

[0525]

[0576] In some embodiments, the serum supplement or serum substitute includes, but is not limited to, one or more CTS(™) OpTmizer(™) T Cell Expansion Serum Supplements, CTS(™) Immune Cell Serum Substitute, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium includes albumin, and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and a trace element moiety Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3 ”, Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ and Zr 4+ and one or more components selected from the group consisting of compounds containing the same. In some embodiments, the defined medium further includes L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0526]

[0577] In some embodiments, the CTS(Trademark) OpTmizer(Trademark) T cell immune cell serum substitute is used with conventional growth media including, but not limited to, CTS(Trademark) OpTmizer(Trademark) T cell growth basal medium, CTS(Trademark) OpTmizer(Trademark) T cell growth SFM, CTS(Trademark) AIM-V medium, CST(Trademark) AIM-V SFM, LymphoONE(Trademark) T cell growth xeno-free medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI growth medium, Iscove's Modified Dulbecco Medium.

[0527]

[0578] In some embodiments, the total serum substitute concentration (volume %) in the serum-free or defined medium is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the total volume of the serum-free or defined medium. In some embodiments, the total serum substitute concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum substitute concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum substitute concentration is about 10% of the total volume of the serum-free or defined medium.

[0528]

[0579] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed prior to use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0529]

[0580] In some embodiments, the defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) together with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and further contains IL-2 at about 1000 IU / mL to about 8000 IU / mL. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and further contains about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol and 2 mM L-glutamine, and further contains about 6000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ ImmuneCell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ ImmuneCell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ ImmuneCell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further contains from about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ ImmuneCell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains from about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ ImmuneCell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ ImmuneCell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further contains about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ ImmuneCell Serum Replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0530]

[0581] In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX™) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX™) at a concentration of 2 mM.

[0531]

[0582] 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 55 mM.

[0532]

[0583] In some embodiments, the defined medium described in International Publication No. WO 1998 / 030679, which is incorporated herein by reference, is useful in the present invention. That publication describes a serum-free eukaryotic cell culture medium. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics, or is obtained by combining them. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and / or β-mercaptoethanol. In some embodiments, the defined medium comprises an albumin or albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag + 、Al 3+ 、Ba 2+ 、Cd 2+ 、Co 2+ 、Cr 3 ”, Ge 4+ 、Se 4+ 、Br、T、Mn 2+ 、P、Si 4+ 、V 5+ 、Mo 6+ 、Ni 2+ 、Rb + 、Sn2+ and Zr 4+ and one or more components selected from the group consisting of compounds containing 4+ . In some embodiments, the basal cell medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Alpha Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, Iscove's Modified Dulbecco's Medium.

[0533]

[0584] In some embodiments, the concentration of glycine in the defined medium ranges from about 5 to 200 mg / L, the concentration of L-histidine is about 5 to 250 mg / L, the concentration of L-isoleucine is about 5 to 300 mg / L, the concentration of L-methionine is about 5 to 200 mg / L, the concentration of L-phenylalanine is about 5 to 400 mg / L, the concentration of L-proline is about 1 to 1000 mg / L, the concentration of L-hydroxyproline is about 1 to 45 mg / L, the concentration of L-serine is about 1 to 250 mg / L, the concentration of L-threonine is about 10 to 500 mg / L, the concentration of L-tryptophan is about 2 to 110 mg / L, the concentration of L-tyrosine is about 3 to 175 mg / L, the concentration of L-valine is about 5 to 500 mg / L, the concentration of thiamine is about 1 to 20 mg / L, the concentration of reduced glutathione is about 1 to 20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1 to 200 mg / L, the concentration of iron-saturated transferrin is about 1 to 50 mg / L, the concentration of insulin is about 1 to 100 mg / L, the concentration of sodium selenite is about 0.000001 to 0.0001 mg / L, and the concentration of albumin (such as AlbuMAX® I) is about 5000 to 50,000 mg / L.

[0534]

[0585] In some embodiments, the non-trace element sub-components in the defined medium are present in the concentration ranges listed in the column under the heading "Concentration Range in 1X Medium" of Table A2 below. In other embodiments, the non-trace element sub-components in the defined medium are present at the final concentrations listed in the column under the heading "Preferred Embodiments of 1X Medium" of Table A2 below. In other embodiments, the defined medium is a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-trace element sub-components of the types and concentrations listed in the column under the heading "Preferred Embodiments of Supplements" of Table A2 below.

[0535]

Table 7

[0536]

[0586] In some embodiments, the volumetric osmolarity of the defined medium is from about 260 to 350 mOsmol. In some embodiments, the volumetric osmolarity is from about 280 to 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g / L or about 2.2 g / L of sodium bicarbonate. The defined medium can be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0537]

[0587] In one embodiment, 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) is useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

[0538]

[0588] In one embodiment, the cell culture medium in the first and / or second gas-permeable container is not filtered. The use of unfiltered cell culture medium can simplify the procedures necessary to expand the cell number. In one embodiment, the cell culture medium in the first and / or second gas-permeable container lacks beta-mercaptoethanol (BME or βME; 2-mercaptoethanol, also known as CAS 60-24-2).

[0539]

[0589] In one embodiment, a second rapid expansion culture (including the expansion culture referred to as REP) is performed, which further includes the step of selecting TILs for excellent tumor responsiveness. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1, the disclosure of which is incorporated herein by reference, can be used for the selection of TILs for excellent tumor reactivity.

[0540]

[0590] Optionally, after the second rapid expansion culture (including the expansion culture referred to as REP expansion culture), a cell viability assay can be performed using standard assays known in the art. For example, a trypan blue dye exclusion assay can be performed on a sample of bulk TIL, which selectively labels dead cells to enable the evaluation of viability. In some embodiments, a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA) can be used to count the TIL samples and determine viability. In some embodiments, viability is determined according to the standard Cellometer K2 Image Cytometer automated cell counter protocol.

[0541]

[0591] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene segments V (variable), D (diversity), J (joining), and C (constant) determine the binding specificity and downstream application of immunoglobulins and T cell receptors (TCRs). The present invention provides a method for generating tumor-infiltrating lymphocytes (TILs) that exhibit and increase the diversity of the T cell repertoire. In some embodiments, the TILs obtained by this method exhibit an increase in T cell repertoire diversity. In some embodiments, the TILs obtained in a second expansion culture exhibit an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in the diversity of immunoglobulins and / or the diversity of T cell receptors. In some embodiments, the diversity of immunoglobulins is in the immunoglobulin heavy chain. In some embodiments, the diversity of immunoglobulins is in the immunoglobulin light chain. In some embodiments, the diversity is in the T cell receptor. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, the expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, the expression of T cell receptor (TCR) alpha is increased. In some embodiments, the expression of T cell receptor (TCR) beta is increased. In some embodiments, the expression of TCRab (i.e., TCRα / β) is increased.

[0542]

[0592] In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as will be discussed in more detail below. In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains, as will be discussed in more detail below, 6000 IU / mL of IL-2, 30 μg / flask of OKT-3, and 7.5×10 8It contains individual antigen-presenting feeder cells (APCs). In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as will be discussed in more detail below. In some embodiments, the second rapid expansion culture medium (e.g., CM2 or sometimes referred to as the second cell culture medium) contains 6000 IU / mL of IL-2, 30 μg / flask of OKT-3, and 5×10 8 individual antigen-presenting feeder cells (APCs).

[0543]

[0593] In some embodiments, the second rapid expansion culture, e.g., step D according to FIG. 1 (particularly FIGS. 1B and / or 1C), is carried out in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL expansion culture. In some embodiments, a bioreactor is used. In some embodiments, the bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-100 or G-REX-500. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-500.

[0544] 1. Feeder Cells and Antigen-Presenting Cells

[0594] In one embodiment, the second rapid expansion culture procedure described herein (e.g., the expansion culture described in step D of FIG. 1 (particularly FIGS. 1B and / or 1C) and the expansion culture referred to as REP) requires an excess amount of feeder cells during REP TIL expansion culture and / or during the second rapid expansion culture. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard unit of whole blood from a healthy donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

[0545]

[0595] Generally, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the REP procedure as described in the examples providing exemplary protocols for assessing the replicative ability of allogeneic PBMCs.

[0546]

[0596] In some embodiments, if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells seeded into culture on day 0 of the REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture), the PBMCs are considered non-replicative and may be used in the TIL expansion culture procedures described herein.

[0547]

[0597] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 has not increased on days 7 and 14 from the initial number of viable cells seeded into culture on day 0 of the REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture), the PBMCs are considered non-replicative and may be used in the TIL expansion culture procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 3000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 60 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 60 ng / mL of OKT3 antibody and 3000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0548]

[0598] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 does not increase on day 7 and day 14 from the initial number of viable cells transferred to culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture), the PBMCs are considered non-replicating and may be approved for use in the TIL expansion culture procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 1000 - 6000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 2000 - 5000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 2000 - 4000 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 2500 - 3500 IU / mL of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30 - 60 ng / mL of OKT3 antibody and 6000 IU / mL of IL-2.

[0549]

[0599] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TIL to antigen-presenting feeder cells in the second expansion culture is about 1:10, about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or about 1:500. In one embodiment, the ratio of TIL to antigen-presenting feeder cells in the second expansion culture is 1:50 - 1:300. In one embodiment, the ratio of TIL to antigen-presenting feeder cells in the second expansion culture is 1:100 - 1:200.

[0550]

[0600] In one embodiment, the second expansion culture procedure described herein is about 5×10 8 feeder cells and about 100×10 6The ratio of individual TILs is required. In one embodiment, the second expansion culture procedure described herein is about 7.5×10 8 individual feeder cells and about 100×10 6 individual TILs. In another embodiment, the second expansion culture procedure described herein is about 5×10 8 individual feeder cells and about 50×10 6 individual TILs. In another embodiment, the second expansion culture procedure described herein is about 7.5×10 8 individual feeder cells and about 50×10 6 individual TILs. In yet another embodiment, the second expansion culture procedure described herein requires from about 5×10 8 individual feeder cells to about 25×10 6 individual TILs. In yet another embodiment, the second expansion culture procedure described herein requires about 7.5×10 8 individual feeder cells and about 25×10 6 individual TILs. In yet another embodiment, the second rapid expansion culture requires twice the number of feeder cells of the second rapid expansion culture. In yet another embodiment, if the first initial stimulation expansion culture described herein requires about 2.5×10 8 individual feeder cells, the second rapid expansion culture requires about 5×10 8 individual feeder cells. In yet another embodiment, if the first initial stimulation expansion culture described herein requires about 2.5×10 8 individual feeder cells, the second rapid expansion culture requires about 7.5×10 8 individual feeder cells. In yet another embodiment, the second rapid expansion culture requires 2 times (2.0X), 2.5X, 3.0X, 3.5X or 4.0X the number of feeder cells of the first initial stimulation expansion culture.

[0551]

[0601] In one embodiment, the second rapid expansion culture procedure described herein requires an excess amount of feeder cells during the second rapid expansion culture. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic healthy donor. 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. In some embodiments, PBMCs are added to the second rapid expansion culture at twice the concentration of PBMCs added to the first priming expansion culture.

[0552]

[0602] Generally, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the TIL expansion culture procedures described herein, including the exemplary procedures described in the figures and examples.

[0553]

[0603] In one embodiment, artificial antigen-presenting cells are used in the second rapid expansion culture as an alternative to or in combination with PBMCs.

[0554] 2. Cytokines

[0604] The second rapid expansion culture method described herein generally uses a culture medium containing high doses of cytokines, particularly IL-2, as is known in the art.

[0555]

[0605] Alternatively, it is further possible to use a combination of cytokines for the second rapid expansion culture of TILs, and for combinations of two or more of IL-2, IL-15, and IL-21, it is generally as outlined in International Publication No. WO 2015 / 189356 and International Publication No. WO 2015 / 189357 (which are hereby expressly incorporated by reference in their entireties). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, and IL-15 and IL-21, with the latter finding particular use in many embodiments. As described in these specifications, using combinations of cytokines is advantageous, particularly for the generation of lymphocytes, specifically T cells.

[0556] E. Step E: Recovery of TILs

[0606] After the second rapid expansion culture step, the cells can be recovered. In some embodiments, the TILs are recovered after 1, 2, 3, 4 or more expansion culture steps, as provided, for example, in FIG. 1 (particularly FIGS. 1B and / or 1C). In some embodiments, the TILs are recovered after two expansion culture steps, as provided, for example, in FIG. 1 (particularly FIGS. 1B and / or 1C). In some embodiments, the TILs are recovered after two expansion culture steps (one first priming expansion culture and one second rapid expansion culture), as provided, for example, in FIG. 1 (particularly FIGS. 1B and / or 1C).

[0557]

[0607] The TILs can be recovered by any suitable and sterile method, including, for example, by centrifugation. TIL recovery methods are well known in the art, and any such known method can be used with this process. In some embodiments, the TILs are recovered using an automated system.

[0558]

[0608] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Optionally, a cell-based harvester can be used in the method. In some embodiments, the cell harvester and / or cell processing system is a membrane-based cell harvester. In some embodiments, cell recovery is performed via a cell processing system such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any device or apparatus manufactured by any vendor that can deliver a cell-containing solution through a membrane or filter, such as a rotating membrane or rotating filter, and remove the supernatant or cell culture medium without pelleting in a sterile and / or closed-system environment, enabling continuous flow and cell processing. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0559]

[0609] In some embodiments, the second rapid expansion culture, for example, step D according to FIG. 1 (particularly, for example, FIGS. 1B and / or 1C), is performed in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for the TIL expansion culture. In some embodiments, a bioreactor is used. In some embodiments, the bioreactor is used as a container. In some embodiments, the bioreactor used is, for example, G-REX-100 or G-REX-500. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-500.

[0560]

[0610] In some embodiments, step E according to FIG. 1 (in particular, for example, FIGS. 1B and / or 1C) is carried out according to the process described herein. In some embodiments, to maintain the sterility and closure of the system, the closed system is accessed via a syringe under sterile conditions. In some embodiments, a closed system as described herein is used.

[0561]

[0611] In some embodiments, TIL is harvested according to the method described herein. In some embodiments, TIL is harvested on days 14 to 16 using a method as described herein. In some embodiments, TIL is harvested on day 14 using a method as described herein. In some embodiments, TIL is harvested on day 15 using a method as described herein. In some embodiments, TIL is harvested on day 16 using a method as described herein.

[0562] F. Step F: Transfer to the final formulation / infusion bag

[0612] After steps A to E have been completed as provided in the exemplary order of FIG. 1 (in particular, for example, FIGS. 1B and / or 1C) and as described above and in detail herein, the cells are transferred to a container for use in administration to a patient. In some embodiments, when a therapeutically sufficient number of TILs are obtained using the expansion culture method described above, the TILs are transferred to a container for use in administration to a patient.

[0563]

[0613] In one embodiment, TILs expanded using the method of the present disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded as disclosed herein can be administered by any suitable route known in the art. In some embodiments, the TILs are administered as a single intra-arterial or intravenous infusion, which is preferably continued for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0564] G.PBMC feeder cell ratio

[0614] In some embodiments, the culture medium used in the expansion culture methods described herein (see, e.g., FIG. 1, particularly FIGS. 1B and / or 1C) includes an anti-CD3 antibody, such as OKT-3. When an anti-CD3 antibody is used in combination with IL-2, T cell activation and cell division are induced in the TIL population. This effect can be seen with full-length antibodies as well as Fab and F(ab’)2 fragments, with the former generally being preferred; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719 (incorporated herein by reference in its entirety).

[0565]

[0615] In one embodiment, the number of PBMC feeder layers is calculated as follows. A. Volume of T cell (diameter 10μm): V = (4 / 3)πr 3 = 523.6μm 3 B. Cylindrical flask of G-Rex 100(M) with a height of 40μm (4 cells): V = (4 / 3)πr 3 = 4×10 12 μm 3 C. Number of cells required to fill cylindrical flask B: 4×10 12 μm 3 / 523.6μm 3 = 7.6×10 8 μm 3 *0.64 = 4.86×10 8 D. Number of cells that can be optimally activated in 4D space: 4.86×10 8 / 24 = 20.25×10 6 E. Number of feeder and TIL extrapolated to G-Rex 500: TIL: 100×10 6 and feeder: 2.5×10 9 In this calculation, 100cm 2An estimate of the number of mononuclear cells required to provide an icosahedral geometry for activating TILs within the base cylinder is used. This calculation yields experimental results of approximately 5×10 8 for the threshold activation of T cells that closely reflect the experimental data of the NCI. (1) (C) Multiplier (0.64) is the random data density of equivalent spheres calculated by Jaeger and Nagel in 1992 (2) . (D) Divisor 24 is the "Newton number" in four-dimensional space (3) and is the number of equivalent spheres that can potentially contact similar objects. (1) Jin, Jianjian, et.al., Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes (TIL) in Gas-Permeable Flasks to Numbers Needed for Patient Treatment. J Immunother. 2012 Apr; 35(3):283-292. (2) Jaeger HM, Nagel SR. Physics of the granular state. Science. 1992 Mar 20; 255(5051):1523-31. (3) O. R. Musin (2003). “The problem of the twenty-five spheres”. Russ. Math. Surv. 58 (4):794-795.

[0566]

[0616] In one embodiment, the number of antigen-presenting feeder cells exogenously supplied during the first priming expansion culture is approximately half the number of antigen-presenting feeder cells exogenously supplied during the second rapid expansion culture. In certain embodiments, the method includes performing the first priming expansion culture in a cell culture medium containing approximately 50% fewer antigen-presenting cells compared to the cell culture medium of the second rapid expansion culture.

[0567]

[0617] In another embodiment, the number of antigen-presenting feeder cells (APCs) exogenously supplied during the second rapid expansion culture is greater than the number of APCs exogenously supplied during the first primary stimulation expansion culture.

[0568]

[0618] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first primary stimulation expansion culture is in the range of 1.1:1 or about 1.1:1 to 20:1 or about 20:1.

[0569]

[0619] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first primary stimulation expansion culture is in the range of 1.1:1 or about 1.1:1 to 10:1 or about 10:1.

[0570]

[0620] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first primary stimulation expansion culture is in the range of 1.1:1 or about 1.1:1 to 9:1 or about 9:1.

[0571]

[0621] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first primary stimulation expansion culture is in the range of 1.1:1 or about 1.1:1 to 8:1 or about 8:1.

[0572]

[0622] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first primary stimulation expansion culture is in the range of 1.1:1 or about 1.1:1 to 7:1 or about 7:1.

[0573]

[0623] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first primary stimulation expansion culture is in the range of 1.1:1 or about 1.1:1 to 6:1 or about 6:1.

[0574]

[0624] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 5:1 or about 5:1.

[0575]

[0625] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 4:1 or about 4:1.

[0576]

[0626] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 3:1 or about 3:1.

[0577]

[0627] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.9:1 or about 2.9:1.

[0578]

[0628] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.8:1 or about 2.8:1.

[0579]

[0629] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.7:1 or about 2.7:1.

[0580]

[0630] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.6:1 or about 2.6:1.

[0581]

[0631] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.5:1 or about 2.5:1.

[0582]

[0632] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.4:1 or about 2.4:1.

[0583]

[0633] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.3:1 or about 2.3:1.

[0584]

[0634] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.2:1 or about 2.2:1.

[0585]

[0635] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2.1:1 or about 2.1:1.

[0586]

[0636] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or in the range of about 1.1:1 to 2:1 or about 2:1.

[0587]

[0637] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first initial stimulation expansion culture is 2:1 or in the range of about 2:1 to 10:1 or about 10:1.

[0588]

[0638] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first initial stimulation expansion culture is 2:1 or in the range of about 2:1 to 5:1 or about 5:1.

[0589]

[0639] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first initial stimulation expansion culture is 2:1 or in the range of about 2:1 to 4:1 or about 4:1.

[0590]

[0640] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first initial stimulation expansion culture is 2:1 or in the range of about 2:1 to 3:1 or about 3:1.

[0591]

[0641] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first initial stimulation expansion culture is 2:1 or in the range of about 2:1 to 2.9:1 or about 2.9:1.

[0592]

[0642] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first initial stimulation expansion culture is 2:1 or in the range of about 2:1 to 2.8:1 or about 2.8:1.

[0593]

[0643] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or in the range of about 2:1 to 2.7:1 or about 2.7:1.

[0594]

[0644] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or in the range of about 2:1 to 2.6:1 or about 2.6:1.

[0595]

[0645] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or in the range of about 2:1 to 2.5:1 or about 2.5:1.

[0596]

[0646] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or in the range of about 2:1 to 2.4:1 or about 2.4:1.

[0597]

[0647] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or in the range of about 2:1 to 2.3:1 or about 2.3:1.

[0598]

[0648] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or in the range of about 2:1 to 2.2:1 or about 2.2:1.

[0599]

[0649] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or in the range of about 2:1 to 2.1:1 or about 2.1:1.

[0600]

[0650] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 2:1 or about 2:1.

[0601]

[0651] In another embodiment, the ratio of the number of APCs exogenously supplied during the second rapid expansion culture to the number of APCs exogenously supplied during the first priming expansion culture is 1.1:1 or about 1.1:1, 1.2:1 or about 1.2:1, 1.3:1 or about 1.3:1, 1.4:1 or about 1.4:1, 1.5:1 or about 1.5:1, 1.6:1 or about 1.6:1, 1.7:1 or about 1.7:1, 1.8:1 or about 1.8:1, 1.9:1 or about 1.9:1, 2:1 or about 2:1, 2.1:1 or about 2.1:1, 2.2:1 or about 2.2:1, 2.3:1 or about 2.3:1, 2.4:1 or about 2.4:1, 2.5:1 or about 2.5:1, 2.6:1 or about 2.6:1, 2.7:1 or about 2.7:1, 2.8:1 or about 2.8:1, 2.9:1 or about 2.9:1, 3:1 or about 3:1, 3.1:1 or about 3.1:1, 3.2:1 or about 3.2:1, 3.3:1 or about 3.3:1, 3.4:1 or about 3.4:1, 3.5:1 or about 3.5:1, 3.6:1 or about 3.6:1, 3.7:1 or about 3.7:1, 3.8:1 or about 3.8:1, 3.9:1 or about 3.9:1, 4:1 or about 4:1, 4.1:1 or about 4.1:1, 4.2:1 or about 4.2:1, 4.3:1 or about 4.3:1, 4.4:1 or about 4.4:1, 4.5:1 or about 4.5:1, 4.6:1 or about 4.6:1, 4.7:1 or about 4.7:1, 4.8:1 or about 4.8:1, 4.9:1 or about 4.9:1, or 5:1 or about 5:1.

[0602]

[0652] In another embodiment, the number of APCs exogenously supplemented during the first priming expansion culture is 1×10 8 or about 1×10 8 、1.1×10 8or about 1.1×10 8 、1.2×10 8 or about 1.2×10 8 、1.3×10 8 or about 1.3×10 8 、1.4×10 8 or about 1.4×10 8 、1.5×10 8 or about 1.5×10 8 、1.6×10 8 or about 1.6×10 8 、1.7×10 8 or about 1.7×10 8 、1.8×10 8 or about 1.8×10 8 、1.9×10 8 or about 1.9×10 8 、2×10 8 or about 2×10 8 、2.1×10 8 or about 2.1×10 8 、2.2×10 8 or about 2.2×10 8 、2.3×10 8 or about 2.3×10 8 、2.4×...

Claims

1. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) performing a first primary stimulation expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a second TIL population, wherein the first primary stimulation expansion culture is performed in a container containing a first gas-permeable surface, the first primary stimulation expansion culture is performed over a first period of 1 to 7 days to obtain the second TIL population, and the second TIL population is greater in number than the first TIL population; (c) performing a second rapid expansion culture by supplementing the cell culture medium of the second TIL population with IL-2, OKT-3, and APCs to generate a third TIL population, wherein the number of APCs added in the second rapid expansion culture is at least twice the number of APCs added in step (b), the second rapid expansion culture is performed over a second period of 1 to 11 days to obtain the third TIL population, the third TIL population is a therapeutic TIL population, and the second rapid expansion culture is performed in a container containing a second gas-permeable surface; (d) recovering the therapeutic TIL population obtained from step (c); (e) engineering the TILs to express an orthogonal IL-2Rβ; and (f) transferring the recovered and engineered TIL population into an infusion bag A method comprising the above steps.

2. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) performing a first primary stimulation expansion culture by culturing the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a second TIL population, wherein the first primary stimulation expansion culture is performed over a first period of 1 to 7 days to obtain the second TIL population, and the second TIL population is greater in number than the first TIL population; (c) performing a second rapid expansion culture by contacting said second TIL population with a cell culture medium comprising orthogonality IL-2, OKT-3, and APC to generate a third TIL population, wherein said second rapid expansion culture is performed over a second period of 1 to 11 days to obtain said third TIL population, and said third TIL population is a therapeutic TIL population; (d) engineering said TIL to express orthogonality IL-2Rβ; and (e) recovering said therapeutic TIL population obtained from step (d) A method comprising.

3. A method for expanding tumor-infiltrating lymphocytes (TIL) into a therapeutic TIL population, (a) culturing a first TIL population, which can be obtained by processing a tumor sample from a tumor excised from a subject into a plurality of tumor fragments, in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APC), to perform a first priming expansion culture to generate a second TIL population, wherein said first priming expansion culture is performed in a container comprising a first gas-permeable surface, said first priming expansion culture is performed over a first period of 1 to 7 days to obtain said second TIL population, and said second TIL population is greater in number than said first TIL population; (b) performing a second rapid expansion culture by contacting said second TIL population with a cell culture medium of said second TIL population having additional IL-2, OKT-3, and APC to generate a third TIL population, wherein the number of APCs in said second rapid expansion culture is at least twice the number of APCs in step (a), said second rapid expansion culture is performed over a second period of 1 to 11 days to obtain said third TIL population, said third TIL population is a therapeutic TIL population, and said second rapid expansion culture is performed in a container comprising a second gas-permeable surface; (c) recovering said therapeutic TIL population obtained from step (b); and (d) engineering said TIL generated in step (c) to express orthogonality IL-2Rβ A method comprising.

4. A method for expanding tumor-infiltrating lymphocytes (TIL) into a therapeutic TIL population, Performing a first primary stimulation expansion culture by culturing a first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a second TIL population, wherein the first primary stimulation expansion culture is performed over a first period of 1 to 7 days to obtain the second TIL population, and the second TIL population is greater in number than the first TIL population; Performing a second rapid expansion culture by contacting the second TIL population with a cell culture medium containing IL-2, OKT-3, and APCs to generate a third TIL population, wherein the second rapid expansion culture is performed over a second period of 1 to 11 days to obtain the third TIL population, and the third TIL population is a therapeutic TIL population; Recovering the therapeutic TIL population obtained from step (b); and Engineering the TILs generated in step (c) to express an orthogonal IL-2Rβ A method comprising the above steps.

5. (i) The ratio of the number of APCs in the second rapid expansion culture to the number of APCs in the first primary stimulation expansion culture is selected from the range of 1.5:1 to 20:1, or (ii) The ratio is selected from the range of 2:1 to 5:1, or (iii) The ratio is selected from the range of 2:1 to 3:1, or (iv) The ratio is 2:

1. (v) The number of APCs in the first initial stimulation expansion culture is 1.0 × 10 6 APCs / cm 2 to 4.5 × 10 6 APCs / cm 2 The method according to claim 1, 2 or 4, selected from the range of.

6. (i) 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, or (ii) 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, or (iii) 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, or (iv) 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, or (v) 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, or (vi) The second TIL population is at least 50 times greater in number than the first TIL population. The method according to any one of claims 1 to 5.

7. The method according to any one of claims 2 to 4, comprising, after the step of collecting the therapeutic TIL population, performing an additional step of transferring the collected therapeutic TIL population to a transfusion bag.

8. The plurality of tumor fragments are distributed in a plurality of separate containers, and in each of the separate containers, the second TIL population is obtained from the first TIL population in the step of the first primary stimulation expansion culture, and the third TIL population is obtained from the second TIL population in the step of the second rapid expansion culture, and the therapeutic TIL population obtained from the third TIL population is collected from each of the plurality of containers and combined to yield the collected TIL population. The method according to any one of claims 2 to 7.

9. The method according to any one of claims 2 to 7, wherein the plurality of tumor fragments are distributed in a single container.

10. In the step of the first primary stimulation expansion culture, the cell culture medium contains antigen-presenting cells (APCs), and the APCs are laminated on the first gas-permeable surface with an average thickness of 1 to 3 cell layers. The method according to claim 8 or 9.

11. In the step of the first primary stimulation expansion culture, the APCs are on the first gas-permeable surface, (i) 1.5 to 2.5 cell layers, or (ii) 2 cell layers, laminated with an average thickness of. The method according to claim 9.

12. In the step of the first primary stimulation expansion culture, the first primary stimulation expansion culture is carried out in a first container containing a first gas-permeable surface, and in the step of the second rapid expansion culture, the second rapid expansion culture is carried out in a second container containing a second gas-permeable surface. The method according to any one of claims 2 to 7.

13. For each container in which the first primary stimulation expansion culture is performed on the first TIL population, the second rapid expansion culture is performed in the same container on the second TIL population generated from the first TIL population. The method according to any one of claims 2 to 11.

14. For each container in which the first primary stimulation expansion culture is performed on the first TIL population in the step of the first primary stimulation expansion culture, the first container includes a first surface, the cell culture medium includes antigen-presenting cells (APCs), and the APCs are laminated on the first gas-permeable surface. The ratio of the average number of layers of APCs laminated in the step of the first primary stimulation expansion culture to the average number of layers of APCs laminated in the step of the second rapid expansion culture is selected from the range of 1:1.1 to 1:

10. The ratio of the average number of layers of APCs laminated in the step of the first primary stimulation expansion culture to the average number of layers of APCs laminated in the step of the second rapid expansion culture is (i) in the range of 1:1.2 to 1:8; (ii) in the range of 1:1.3 to 1:7; (iii) in the range of 1:1.4 to 1:6; (iv) in the range of 1:1.5 to 1:5; (v) in the range of 1:1.6 to 1:4; (vi) in the range of 1:1.7 to 1:3.5; (vii) in the range of 1:1.8 to 1:3; (viii) in the range of 1:1.9 to 1:2.5; (ix) 1:2, and is selected from the method according to any one of claims 2 to 9, 12 or 13.

15. (i) After 2 to 3 days in the step of the second rapid expansion culture, the cell culture medium is supplemented with additional IL-2 and / or (ii) the method further includes cryopreserving the recovered TIL population in the step of recovering the therapeutic TIL population using a cryopreservation process. The method according to any one of claims 1 to 14.

16. The method according to claim 1 or 7, further including the step of cryopreserving the infusion bag.

17. The cryopreservation process is carried out using a 1:1 ratio of the recovered TIL population to the cryopreservation medium. The method according to claim 15 or 16.

18. (i) The antigen-presenting cells are peripheral blood mononuclear cells (PBMCs), or (ii) In the step of the first initial stimulation expansion culture, the cell culture medium contains peripheral blood mononuclear cells (PBMCs), and the total number of PBMCs added to the cell culture medium in the step of the first initial stimulation expansion culture is 2.5×10 8 cells, and / or (iii) In the step of the second rapid expansion culture, the antigen-presenting cell (APC) in the cell culture medium is a peripheral blood mononuclear cell (PBMC), and the total number of PBMCs added to the cell culture medium in the step of the second rapid expansion culture is 5×10 8 cells, the method according to any one of claims 1 to 17.

19. The recovery in the step of recovering the therapeutic TIL population is (i) a membrane-based cell processing system and / or (ii) a LOVO cell processing system, and is carried out using the method according to any one of claims 1 to 18.

20. (i) In the step of the first initial stimulation expansion culture, the plurality of fragments include 60 fragments per container, and each fragment has a volume of 27 mm 3 or (ii) The plurality of fragments include 30 to 60 fragments having a total volume of 1300 mm 3 to 1500 mm 3 or not (iii) The plurality of fragments include 50 fragments having a total volume of 1350 mm 3 or (iv) The plurality of fragments includes 50 fragments having a total mass of 1 gram to 1.5 grams. The method according to any one of claims 1 to 19.

21. The method according to any one of claims 1 to 20, wherein the IL-2 concentration is from 10,000 IU / mL to 5,000 IU / mL.

22. (i) The first period in the step of the first initial stimulation expansion culture and the second period in the step of the second rapid expansion culture are each individually carried out within a period of 5 days, 6 days or 7 days, or (ii) The first period in the step of the first initial stimulation expansion culture is carried out within a period of 5 days, 6 days or 7 days, or (iii) The second period in the step of the second rapid expansion culture is carried out within a period of 7 days, 8 days or 9 days, or (iv) The first period in the step of the first initial stimulation expansion culture and the second period in the step of the second rapid expansion culture are each individually carried out within a period of 7 days, or (v) The step from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population is carried out within a period of 14 days to 16 days, or (vi) The step from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population is carried out within a period of 15 days to 16 days, or (vii) The step from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population is carried out within a period of 14 days, or (viii) The step from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population is carried out within a period of 15 days, or (ix) The step from the first initial stimulation expansion culture to the recovery of the therapeutic TIL population is carried out within a period of 16 days, or (x) The method further comprises the step of cryopreserving the recovered therapeutic TIL population using a cryopreservation process, and the step from the first initial stimulation expansion culture to the recovery and cryopreservation of the therapeutic TIL population is carried out within 16 days or less. The method according to any one of claims 1 to 21.

23. (i) The recovered therapeutic TIL population recovered in the step of recovering the therapeutic TIL population contains sufficient TIL for a therapeutically effective dose of the TIL, or (ii) The third TIL population in the step of the second rapid expansion culture provides increased efficacy, increased interferon-gamma production and / or increased polyclonality. (iii) The third TIL population in the step of the second rapid expansion culture provides at least 1 to 5 times or more interferon gamma production compared to TIL prepared by a process longer than 18 days, or (iv) The effector T cells and / or central memory T cells obtained from the third TIL population in the step of the second rapid expansion culture exhibit increased CD8 and CD28 expression compared to the effector T cells and / or central memory T cells obtained from the second TIL population in the step of the first priming expansion culture. The method according to any one of claims 1 to 22. [

24. ] (i) The operation for expressing IL-2Rβ is carried out between step (b) and step (c), or (ii) The operation for expressing IL-2Rβ is carried out between step (c) and step (d), or (iii) Orthogonal IL-2 replaces the IL-2 in step (c). (iv) The method according to claim 1 or 2.

Images