Re-stimulation of cryopreserved tumor infiltrating lymphocytes

Abstract

Re-Stimulation of Cryopreserved Tumor Infiltrating Lymphocytes SOLUTION: The present disclosure provides methods of re-stimulating a population of TILs that lead to improved phenotype and increased metabolic health of the TILs, and provides methods of assaying the population of TILs to determine if they are suitable for high efficacy infusion after re-stimulation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application relates to U.S. Provisional Patent Application No. 6, filed October 26, 2016, entitled "Expansion of Tumor-Infiltrating Lymphocytes and Methods of Using the Same." Nos. 2 / 413,283 and 62 / 413,387, filed October 31, 2016, entitled "RESTIMULATION OF CRYOPRESERVED TUMOR INFILTRATING LYMPHOCYTES" This application claims priority to U.S. Provisional Patent Application No. 62 / 415,452 entitled "Compounds for Implantable and Non-Immunocapable Electrodes," which is hereby incorporated by reference in its entirety. [Background technology]

[0002] Background of the Invention

[0002] The treatment of large, refractory cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) represents a powerful therapeutic approach for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires large amounts of TILs, and a robust and reliable method is needed for commercialization. This has been challenging due to the technical, logistical, and regulatory challenges associated with cell expansion. IL-2-based TIL expansion followed by the "rapid expansion process" (REP) has been shown to be a promising approach. Due to its speed and efficiency, it is becoming the preferred method for TIL expansion. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-fold expansion of TILs in a 14-day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs) as feeder cells, often from multiple donors, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2. (Dudley, et al., J. Immunother. 2003, 26, 332-42).

[0003]

[0003] TILs undergoing the REP procedure have produced successful adoptive cell therapy in melanoma patients after host immunosuppression. Current infusion eligibility parameters rely on readings of TIL composition (e.g., CD28, CD8, or CD4 positivity) and the fold expansion and viability of the REP product.

[0004] However, current REP protocols, as well as current TIL expansion and culture protocols in general, provide little insight into the health of the TILs that will be infused into patients. T cells undergo a significant metabolic shift during their maturation from naive to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, expressly incorporated herein in its entirety, and particularly the discussion and markers of anaerobic and aerobic metabolism). For example, naive T cells rely on mitochondrial respiration to produce ATP, whereas mature, healthy effector T cells, such as TILs, are highly glycolytic and rely on aerobic glycolysis to provide the bioenergetic substrates they require for proliferation, migration, activation, and antitumor efficacy.

[0005] Additionally, such expanded cell populations can be cryopreserved, allowing for ease of use, long-term storage for multiple reinfusions into patients with recurrent disease, and other considerations. However, current infusion eligibility parameters rely on readings of TIL composition and the expansion fold and viability of expanded TIL-based products. These measures provide little insight into the health of the TILs that will be infused into patients, and little is known about the impact of cryopreservation on TIL populations. Summary of the Invention [Problem to be solved by the invention]

[0006]

[0006] Thus, the present invention relates to a method for expanding and restimulating a TIL population, leading to improved phenotype and increased metabolic health of the TILs, and to a method for assaying a TIL population after restimulation to determine whether it is suitable for highly effective infusion. [Means for solving the problem]

[0007] Summary of the Invention

[0007] The present invention provides methods for the expansion of TILs in large, sometimes therapeutic, populations, optionally combined with cryopreservation.

[0008] According to the present disclosure, there is provided a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising the steps of: (i) obtaining a first population of TILs from a tumor resected from a patient; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population; wherein the third TIL population is at least 50-fold or 100-fold more numerous than the second TIL population, and the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population that includes an increased effector T cell and / or central memory T cell subpopulation compared to the second TIL population.

[0009]

[0009] In some embodiments, the method comprises: (iv) performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population. wherein an additional second expansion culture is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (iii), wherein the larger therapeutic TIL population comprises increased effector T cell and / or central memory T cell subpopulations relative to the third TIL population.

[0010] In some embodiments, after step (iii), the cells are removed from the cell culture and cryopreserved in a storage medium before performing step (iv).

[0011]

[0011] In some embodiments, the cells are thawed prior to performing step (iv).

[0012] In some embodiments, step (iv) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dose of TILs in the therapeutic TIL population.

[0013] In some embodiments, steps (i)-(iii) or (iv) are performed within a period of about 40 days to about 50 days. In some embodiments, steps (i)-(iii) or (iv) are performed within a period of about 42 days to about 48 days. In some embodiments, steps (i)-(iii) or (iv) are performed within a period of about 42 days to about 45 days. In some embodiments, steps (i)-(iii) or (iv) are performed within about 44 days.

[0014] In some embodiments, the cells from step (iii) or (iv) are They express D4, CD8, and TCRαβ to the same extent as freshly harvested cells.

[0015] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture in step (iii) on any day between 9 and 17.

[0016]

[0016] In some embodiments, the effector T cells and / or central memory T cells in the therapeutic TIL population of step (iv) exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0017] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

[0018] In some embodiments, the APC is an artificial APC (aAPC).

[0019] In some embodiments, the method further comprises transducing the first TIL population with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

[0020]

[0020] In some embodiments, the method further includes a step of transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

[0021] In some embodiments, the therapeutic TIL population is infused into the patient.

[0022] In some embodiments, step (iii) further comprises removing the cells from the cell culture medium.

[0023] In some embodiments, step (iii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dosage of TILs in the therapeutic TIL population.

[0024] In some embodiments, the number of TILs sufficient to provide a therapeutically effective dose is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0025]

[0025] The present disclosure also provides an expanded cultured TIL population produced by the method of claim 1.

[0026]

[0026] The present disclosure also provides an expanded cultured TIL population produced by the method of claim 1, wherein the expanded cultured TILs have at least a two-fold increase in basal glycolysis when compared to thawed cryopreserved TILs.

[0027]

[0027] The present disclosure also provides methods for assessing the metabolic activity of TIL cell populations generated by the methods described herein, which include measuring the basal glycolysis of the cells.

[0028]

[0028] The present disclosure also provides methods for assessing the metabolic activity of TIL cell populations generated by the methods described herein, which include measuring the basal respiration of the cells.

[0029] The present disclosure also provides a method for the production of TIL cell populations produced by the methods described herein. Also provided is a method for assessing metabolic activity, which involves measuring the spare respiratory capacity (SRC) of cells.

[0030]

[0030] The present disclosure also provides methods for assessing the metabolic activity of TIL cell populations generated by the methods described herein, which include measuring the glycolytic reserve of the cells.

[0031] The present disclosure also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) performing a first expansion culture by culturing a first population of TILs from a tumor resected from a patient in a cell culture medium containing IL-2 to obtain a second population of TILs; and (ii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to obtain a third TIL population, wherein the third TIL population is at least 50-fold or 100-fold more numerous than the second TIL population, and wherein the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population comprising increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population.

[0032] In some embodiments, the method comprises: (iii) further comprising performing an additional second expansion culture of the third TIL population by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (ii), wherein the larger therapeutic TIL population exhibits increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population.

[0033] In some embodiments, the cells from the cell culture medium of step (ii) are removed and cryopreserved in a storage medium prior to step (iii).

[0034] In some embodiments, the cells are thawed prior to step (iii).

[0035]

[0035] In some embodiments, step (ii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dose of TILs in the therapeutic TIL population.

[0036] In some embodiments, the number of TILs sufficient to provide a therapeutically effective dose is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0037] In some embodiments, the APCs are peripheral blood mononuclear cells (PBMCs).

[0038]

[0038] In some embodiments, the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0039] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

[0040] The present disclosure also provides a method of treating a subject having cancer, comprising administering expanded tumor-infiltrating lymphocytes (TILs), the method comprising: (i) Obtaining a first population of TILs from a tumor resected from a patient; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; (iii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold or 100-fold more numerous than the second TIL population, and the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population that includes increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population; and (iv) administering to the patient a therapeutically effective dosage of the third population of TILs. Includes.

[0041]

[0041] In some embodiments, the method further comprises, prior to step (iv), performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (iii), the larger therapeutic TIL population comprising increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population.

[0042]

[0042] In some embodiments, after step (ii), the cells are removed from the cell culture medium and cryopreserved in storage medium prior to an additional second expansion culture according to the methods described herein.

[0043] In some embodiments, the cells are thawed prior to an additional second expansion according to the methods described herein.

[0044] In some embodiments, step (iii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dose of TILs in the therapeutic TIL population.

[0045] In some embodiments, the number of TILs sufficient to provide a therapeutically effective dose is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0046] In some embodiments, the APCs are peripheral blood mononuclear cells (PBMCs).

[0047]

[0047] In some embodiments, the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0048] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

[0049]

[0049] In some embodiments, the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer.

[0050] The present disclosure also provides a method of treating a subject having cancer, comprising administering expanded tumor-infiltrating lymphocytes (TILs), the method comprising: (i) performing a first expansion culture by culturing a first population of TILs from a tumor resected from a patient in a cell culture medium containing IL-2 to obtain a second population of TILs; (ii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to obtain a third TIL population, wherein the third TIL population is at least 50-fold or 100-fold more numerous than the second TIL population, and the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population that includes increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population; and (iii) administering a therapeutically effective dose of the therapeutic TIL population to the patient. Includes.

[0051]

[0051] In some embodiments, the method further comprises, prior to step (iii), performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (ii), wherein the larger therapeutic TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population.

[0052] In some embodiments, the cells from the cell culture medium of step (ii) are removed and cryopreserved in a storage medium prior to an additional second expansion culture as described herein.

[0053] In some embodiments, the cells are thawed prior to an additional second expansion culture as described herein.

[0054] In some embodiments, step (ii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dosage of TILs in the therapeutic TIL population.

[0055] In some embodiments, the number of TILs sufficient to provide a therapeutically effective dose is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0056] In some embodiments, the APCs are peripheral blood mononuclear cells (PBMCs).

[0057]

[0057] In some embodiments, the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0058] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

[0059]

[0059] In some embodiments, the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer.

[0060] The present disclosure also provides an assay method for determining TIL viability. The present disclosure also provides a method for assaying tumor infiltrating lymphocyte (TIL) viability by expanding TIL into a larger TIL population, the method comprising: (i) Obtaining a first population of pre-expanded TILs; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold or 100-fold more numerous than the second TIL population, and wherein the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population, and wherein the third population is further assayed for viability.

[0061] In some embodiments, the method comprises: (iv) performing an additional second expansion by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population; wherein an additional second expansion culture is performed for at least 14 days to obtain a larger TIL population than that obtained in step (iii), the larger TIL population comprising increased effector T cell and / or central memory T cell subpopulations relative to the third TIL population, and the third population is further assayed for viability.

[0062] In some embodiments, prior to step (i), the cells are cryopreserved.

[0063] In some embodiments, the cells are thawed prior to performing step (i).

[0064] In some embodiments, step (iv) is repeated 1 to 4 times to obtain enough TILs for analysis.

[0065] In some embodiments, steps (i) through (iii) or (iv) are carried out within a period of about 40 days to about 50 days.

[0066] In some embodiments, steps (i) through (iii) or (iv) are carried out within a period of about 42 days to about 48 days.

[0067] In some embodiments, steps (i) through (iii) or (iv) are carried out within a period of about 42 days to about 45 days.

[0068] In some embodiments, steps (i)-(iii) or (iv) are performed within about 44 days.

[0069] In some embodiments, the cells from step (iii) or (iv) express CD4, CD8, and TCRαβ to the same extent as freshly harvested cells.

[0070] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

[0071] In some embodiments, the PBMCs are added to the cell culture in step (iii) on any day between 9 and 17.

[0072] In some embodiments, the effector T cells and / or central memory T cells in the larger TIL population of step (iv) have at least one of the following characteristics: CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population. It exhibits one or more selected characteristics.

[0073] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

[0074] In some embodiments, the APC is an artificial APC (aAPC).

[0075]

[0075] In some embodiments, the method further comprises transducing the first TIL population with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

[0076] In some embodiments, a transduction step is performed before step (i).

[0077]

[0077] In some embodiments, the method further comprises transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

[0078] In some embodiments, a transduction step is performed before step (i).

[0079] In some embodiments, the TILs are assayed for viability.

[0080] In some embodiments, TILs are assayed for viability after cryopreservation.

[0081] In some embodiments, the TILs are assayed for viability after cryopreservation and after step (iv).

[0082]

[0082] According to the present disclosure, further uses in methods of assaying the viability of TILs and / or administering them to a subject. In some embodiments, a method of assaying tumor infiltrating lymphocytes (TILs) comprises: (i) Obtaining the first TIL population; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population; (iv) harvesting, washing, and cryopreserving the third TIL population; (v) cryopreserved TILs are stored at cryogenic temperatures; (vi) thawing the third TIL population to provide a thawed third TIL population; and (vii) performing an additional second expansion of a portion of the thawed third TIL population for a reREP period of at least 3 days by adding IL-2, OKT-3, and APC to the cell culture medium of the third population, wherein the third expansion is performed to obtain a fourth TIL population, and comparing the number of TILs in the fourth TIL population with the number of TILs in the third TIL population to determine a ratio; (viii) determining whether the thawed TIL population is suitable for administration to a patient based on the ratio of step (vii); (ix) administering a therapeutically effective dose of the thawed third TIL population to the patient when the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is determined to be greater than 5:1 in step (viii). Includes.

[0083]

[0083] In some embodiments, the reREP period is carried out until the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is greater than 50:1.

[0084] In some embodiments, the number of TILs sufficient to provide a therapeutically effective dose is about 2.3×10 10 ~Approx. 13.7×10 10 There are individuals.

[0085] In some embodiments, steps (i)-(vii) are performed within a period of about 40 days to about 50 days. In some embodiments, steps (i)-(vii) are performed within a period of about 42 days to about 48 days. In some embodiments, steps (i)-(vii) are performed within a period of about 42 days to about 45 days. In some embodiments, steps (i)-(vii) are performed within about 44 days.

[0086] In some embodiments, the cells from step (iii) or (vii) express CD4, CD8, and TCRαβ to the same extent as freshly harvested cells. In some embodiments, the cells are TILs.

[0087] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture in step (iii) on any day between 9 and 17.

[0088]

[0088] In some embodiments, the effector T cells and / or central memory T cells in the larger TIL population of step (iii) or (vii) exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0089] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

[0090] In some embodiments, the APC is an artificial APC (aAPC).

[0091]

[0091] In some embodiments, a step of transducing the first TIL population with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

[0092] In some embodiments, a transduction step is performed before step (i).

[0093]

[0093] In some embodiments, a step of transducing a first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

[0094] In some embodiments, a transduction step is performed before step (i).

[0095] In some embodiments, the TILs are evaluated for viability after step (vii). Assayed.

[0096]

[0096] The present disclosure also provides additional methods for assaying TILs. In some embodiments, the present disclosure provides methods for assaying TILs, the methods comprising: (i) Obtaining a portion of the first cryopreserved TIL population; (ii) thawing a portion of the first cryopreserved TIL population; (iii) performing a first expansion culture by culturing a portion of the first TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for a reREP period of at least 3 days to generate a second TIL population, wherein the portion from the first TIL population is compared with the second TIL population to determine a ratio of the number of TILs, and the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to a patient based on the ratio of step (iii); (v) determining that the first TIL population is suitable for use in therapeutic administration when the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1 in step (iv). Includes.

[0097] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 50:1.

[0098]

[0098] In some embodiments, the method further comprises performing expansion culture of the entire first cryopreserved TIL population from step (i) by a method as described in any embodiment provided herein.

[0099] In some embodiments, the method further comprises administering the entire first cryopreserved TIL population from step (i) to the patient.

[0100] The present disclosure also provides additional methods for assaying TILs. The present disclosure provides a method for assaying TILs, the method comprising: (i) Obtaining a portion of the first cryopreserved TIL population; (ii) thawing a portion of the first cryopreserved TIL population; (iii) performing a first expansion culture by culturing a portion of the first TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for a reREP period of at least 3 days to generate a second TIL population, wherein the portion from the first TIL population is compared with the second TIL population to determine a ratio of the number of TILs, and the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to the patient based on the ratio of step (iii); and (v) when the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1 in step (iv), therapeutically administering the remainder of the first TIL population to the patient. Includes.

[0101] In some embodiments, the number of TILs in the second TIL population is compared to the number of TILs in the first TIL population. The ratio of TIL numbers in some groups is greater than 50:1.

[0102]

[0102] In some embodiments, the method comprises the steps of any of the methods of the preceding claims. and performing an expansion culture of the entire first cryopreserved TIL population from step (i).

[0103] In some embodiments, the method further comprises the step of: It further includes administering the entire TIL population to the patient.

[0104] In some embodiments, the method comprises assessing the metabolic health of the second TIL population. The method further includes the step of:

[0105] In some embodiments, the method comprises assessing the phenotype of the second TIL population. The method further includes the steps of:

[0106] In some embodiments, the antigen-presenting cells are allogeneic peripheral blood mononuclear cells. [Brief explanation of the drawings]

[0107] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[0107] Results from Example 1 are shown. As this table shows, after antigen restimulation rapid expansion protocol ("reREP"), TILs exhibit a significant enhancement of their glycolytic respiration. SRC = spare respiratory capacity. [Figure 2]

[0108] Composition of fresh and thawed TILs. TILs were stained for TCRαβ and CD56 to define T cell and NK populations. Data shown are the average of six individual TILs. [Figure 3]

[0109] The memory phenotype is defined by CD45RA and CCR7 expression. CD4 and CD8 TILs are predominantly effector memory (EM). This remains the same in thawed TILs. Each point represents one sample analyzed. No significant differences were observed by Wilcoxon paired signed-rank test. [Figure 4]

[0110] Pearson correlation of CD4, CD8, CD4+CD28+, and CD8+CD28+ frequencies between fresh and thawed TILs. Cells were stained with the markers listed above. Each point represents a single cell, with fresh values ​​on the x-axis and thawed values ​​on the y-axis. Fitting lines were drawn using linear regression analysis. [Figure 5]

[0111] Comparable activation markers for fresh and thawed TILs. No significant differences in activation status were observed between fresh and thawed TILs using the Wilcoxon paired rank test. Each point represents one sample analyzed and is shown as the mean ± SEM. [Figure 6]

[0112] Maintenance of LAG-3 staining after cryopreservation and thawing. A: LAG-3 staining of CD8 TILs. B: % frequency of regulatory molecules in CD4 and CD8 populations for fresh and thawed TILs. CD8+TIM-3+ and CD8+LAG-3+ thawed TILs have lower % than fresh TILs. Mann-Whitney statistical test. [Figure 7]

[0113] Tumor-infiltrating lymphocytes (TILs) are remarkably stable in their infusion phenotype after cryopreservation. [Figure 8]

[0114] Scatter plot showing phenotypic characterization of reREP TILs: Q1 shows 19.0% CD45RA+ / CCR7-; Q2 shows 0.066% CD45RA+ / CCR7+; Q4 shows 80.6% CD45RA- / CCR7-; and Q3 shows 0.36% CD45RA- / CCR7+. [Figure 9]

[0115] Figures and data showing phenotypic characterization of reREP TILs during the first and second expansion stages. On day 14, after the first expansion but before the second expansion, 0.08% CD45RA+ / CCR7-; 0.03% CD45RA+ / CCR7+; 73.97% CD45RA- / CCR7-; and 25.91% CD45RA- / CCR7+. Proliferation of CM or EM TILs in repeated ReREP. Repeated ReREP was used to test the proliferative potential of central memory (CM) TILs and effector memory (EM) TILs. Briefly, 1.3 × 106 post-REP TILs were cocultured with 1.3 × 107 PBMC feeders (CFSE-labeled), OKT3 (30 ng / nL), and rhIL-2 (3000 IU / mL), and the cultures were incubated for 14 days. On day 14, central memory TILs and effector memory TILs were gated on the L / D Aqua- / CFSE- / TCRα / β+ / CD45RA- / CCR7+ and L / D Aqua- / CFSE- / TCRα / β+ / CD45RA- / CCR7- populations, respectively, and sorted by flow cytometry. The purity of the cell populations was 97%. Next, 1 × 104 flow cytometry-sorted CM or EM or unsorted TILs were cultured in triplicate with 1 × 106 PBMC feeders, OKT3 (30 ng / nL), and IL-2 (3000 IU / ml) for 7 days. Cells were counted and scored. Central memory TILs were more proliferative than effector memory TILs. We are currently repeating this experiment with a larger number of post-REP TIL lines. [Figure 10]

[0116] Figures 10A and 10B: Phenotypic characterization of TILs during ReREP. Cells were gated on Aqua- / TCRα / β+ / CD4+ or CD8+ to reveal central memory TIL (CD45RA-CCR7+) or effector memory TIL (CD45RA-CCR7-) memory phenotypes. Statistical significance was calculated using Student's t. *p<0.05, ns not significant. [Figure 11]

[0117] 1 is an exemplary schematic diagram of a TIL preparation process, sometimes referred to herein as the 1C process. [Figure 12]

[0118] Successful expansion of TILs from non-melanoma tumors. Data show the distribution of TILs (CD4+ / CD8+) in non-melanoma tumors. [Figure 13]

[0119] Non-melanoma TILs expressed CD27 and CD38, consistent with immature TILs. [Figure 14]

[0120] Activated TILs are biased toward effector memory populations. [Figure 15]

[0121] Fresh and reREP TIL phenotypes. DETAILED DESCRIPTION OF THE INVENTION

[0108] Detailed Description of the Invention I. Introduction

[0122] Rapid Expansion Protocol (REP) Adoptive cell therapy utilizing ex vivo cultured TILs has produced successful adoptive cell therapy in melanoma patients following host immunosuppression. Current infusion eligibility parameters rely on readings of TIL composition (e.g., CD28, CD8, or CD4 positivity) and numerical values ​​of fold expansion and survival of the REP product.

[0109]

[0123] The current REP protocol does not guarantee the health of the TILs that will be infused into patients. T cells undergo a significant metabolic shift during their maturation from naive to effector T cells (Chang, et al., Nat. Immunol. 2016, 17, 364 (hereby expressly incorporated in its entirety), and in particular for the discussion and markers of anaerobic and aerobic metabolism.) For example, naive T cells rely on mitochondrial respiration for the production of ATP, while mature, healthy effector T cells, such as TILs, are highly glycolytic and rely on aerobic glycolysis to provide the bioenergetic substrates they require for proliferation, migration, activation, and anti-tumor efficacy.

[0110]

[0124] Previous studies have shown that cells that rely heavily on glycolysis experience nutrient deprivation during adoptive transfer. Therefore, it is desirable to limit glycolysis and promote mitochondrial metabolism in TILs before transplantation because the majority of the transplanted cells will die. Therefore, the art teaches that promoting mitochondrial metabolism may promote in vivo lifespan, and in fact, it has been proposed to use glycolysis inhibitors before inducing an immune response. See Chang et al. (Chang, et al., Nat. Immunol. 2016, 17(364), 574-582).

[0111]

[0125] In a preferred embodiment, the present invention uses freshly harvested TILs or restimulated TILs. Surprisingly, the central memory (CD45RA) TILs were significantly higher in the thawed cryopreserved TILs (sometimes referred to herein as "reTILs") than the thawed cryopreserved TILs. - CCR7 + ) or Effector Memory (CD45RA - CCR7 - The present invention relates to a novel method of enhancing REP with an additional restimulation protocol, sometimes referred to herein as a "re-stimulation rapid expansion protocol" or "reREP," which leads to the expansion of memory T cell subsets, including a .DELTA.) phenotype, and / or leads to a significant enhancement of glycolytic respiration. That is, by using the reREP procedure (i.e., a procedure including a first expansion and a second expansion) on cryopreserved TILs, patients can receive highly metabolically active and healthy TILs, which can lead to better outcomes.

[0112]

[0126] The present invention further provides, in some embodiments, a method for determining and evaluating this increased metabolic health. Accordingly, the present invention provides methods for assaying the relative health of a TIL population using one or more common metabolic determinations, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve.

[0113]

[0127] Moreover, the present invention further provides that, in some embodiments, this increased metabolic health can be achieved by Accordingly, the present invention provides methods for assaying the relative health of a TIL population using one or more common metabolic measures, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve.

[0114]

[0128] Additionally, optional additional determinations include, but are not limited to, ATP production , mitochondrial mass and glucose uptake.

[0115]

[0129] In some cases, reREP cell populations with increased metabolic health are described in the art. The solution is injected into the patient as is generally known in the art.

[0116] II. Definition

[0130] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to cells that migrate through the bloodstream of a subject. "TILs" refers to a population of cells originally obtained as white blood cells that have detached and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations that have been expanded or grown as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein.

[0117]

[0131] TILs can also be generally defined biochemically using cell surface markers. TILs can also be functionally defined by their ability to infiltrate tumors and achieve a therapeutic effect. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may be further characterized by potency—for example, TILs can be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. Interferons can include interferon gamma (IFNγ).

[0118]

[0132] As used herein, "cryopreserved TIL" refers to TILs that have been cultured in primary, bulk, or expanded cultures ( Cryopreservation refers to the processing and storage of TILs, either cryopreserved or cryopreserved (e.g., cryopreserved TILs, cryopreserved TILs, or cryopreserved TILs), at temperatures ranging from about -150°C to -60°C. General cryopreservation methods are also described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs.

[0119]

[0133] As used herein, "thawed cryopreserved TILs" refers to TILs that have been previously cryopreserved. By "TILs" is meant a population of TILs that has been cultured at room temperature and then treated to return to room temperature or above, including but not limited to, cell culture temperature or a temperature at which the TILs may be administered to a patient.

[0120]

[0134] As used herein, a "cell population" (including TILs) refers to a group of cells that share a common trait. refers to a large number of cells. Generally, a population is roughly 1 x 10 6 ~1×10 10 The range is 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion cultures typically yield a bulk TIL population of 1.5 x 10 cells. 9 ~1.5×10 10 This is done to provide a population of cells for injection.

[0121]

[0135] Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then The TILs are expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

[0122]

[0136] Generally, harvested cell suspensions are referred to as "primary cell populations" or "freshly harvested." These are called cell populations.

[0123]

[0137] Generally, as discussed herein, TILs are initially The TILs are prepared by obtaining a primary TIL population from a tumor resected from a patient as described above (the "primary cell population" or "first cell population"), followed by an initial bulk expansion using culturing the cells with IL-2 to form a second cell population (sometimes referred to herein as the "bulk TIL population" or "second population").

[0124]

[0138] The term "cytotoxic lymphocytes" includes cytotoxic T (CTL) cells (CD8 + Cytotoxic T lymphocytes and CD4 + Cytotoxic lymphocytes include T-helper lymphocytes, natural killer T (NKT) cells, and natural killer (NK) cells. Cytotoxic lymphocytes can include, for example, peripheral blood-derived α / β TCR-positive T cells or α / β TCR-positive T cells activated by tumor-associated antigens and / or transduced with tumor-specific chimeric antigen receptors or T cell receptors, and tumor-infiltrating lymphocytes (TILs).

[0125]

[0139] The term "central memory T cells" refers to cells that are CD45RO+ in humans and Central memory T cells are a subset of T cells that constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMII. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are predominant in the CD4 compartment of the blood and are proportionally concentrated in lymph nodes and tonsils in humans.

[0126]

[0140] The term "effector memory T cells" is similar to that of central memory T cells. Human or mammalian T cells that are predominantly CD45R0+ but lack constitutive expression of CCR7 (CCR7lo) and have heterogeneous or low CD62L expression (CD62Llo). The term "closed system" refers to a subset of T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. After antigen stimulation, effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in the blood, and in humans, are proportionally concentrated in the lungs, liver, and intestine. CD8+ effector memory T cells have high amounts of perforin. The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Examples of closed systems include, but are not limited to, closed G containers. After tumor segments are added to the closed system, the system is not opened to the external environment until just before the TILs are administered to the patient.

[0127]

[0141] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to lymphocytes (T cells, B cells, N cells, etc.). It refers to peripheral blood cells with round nuclei, including erythrocytes (K cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0128]

[0142] The term "rapid expansion culture" refers to a culture that expands at least about three-fold (or four-fold, five-fold, or six-fold) over a one-week period. By rapid expansion is meant an increase in the number of antigen-specific TILs by at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a one week period, or most preferably at least about 100-fold over a one week period. Several rapid expansion protocols are described herein.

[0129]

[0143] In some embodiments, the methods of the present disclosure include the step of extracting tumor tissue or cells from tumor tissue. The method further includes a "pre-REP" stage in which cells are grown in standard laboratory media (including, without limitation, RPMI) and treated with reagents such as irradiated feeder cells and anti-CD3 antibodies to achieve a desired effect, such as an increase in TIL numbers and / or enrichment of the population for cells containing desired cell surface markers or other structural, biochemical, or functional characteristics. The pre-REP stage may utilize laboratory-grade reagents (with the understanding that the laboratory-grade reagents will be diluted during the subsequent REP stage) to facilitate the incorporation of alternative strategies for improving TIL production. Thus, in some embodiments, the culture medium during the pre-REP stage may include the disclosed TLR agonists and / or peptides or peptidomimetics. The pre-REP culture may, in some embodiments, include IL-2.

[0130]

[0144] In a preferred embodiment, the present invention uses freshly harvested TILs or restimulated TILs. The present invention relates to a novel method of enhancing REP with an additional restimulation protocol, sometimes referred to herein as a "re-stimulation rapid expansion protocol" or "reREP," which unexpectedly leads to an expansion of memory T cell subsets, including memory effector T cell subsets, and / or leads to a significant enhancement of glycolytic respiration when compared to thawed cryopreserved TILs for cryopreserved TILs (sometimes referred to herein as "reTILs"). That is, by using the reREP procedure on cryopreserved TILs, patients can receive highly metabolically active and healthy TILs, which may lead to better outcomes. Such restimulation protocols, also referred to herein as additional "expansion" of the cell population, are described in further detail herein.

[0131]

[0145] The terms "fragment," "fragment," and "fragmented" refer to the destruction process of a tumor. As used herein to describe processes, the term "in vivo" includes mechanical fragmentation methods such as crushing, slicing, splitting, and mincing tumor tissue, as well as any other method that disrupts the physical structure of tumor tissue. The term "in vivo" refers to events that occur inside a subject's body.

[0132]

[0146] The term "in vitro" refers to events that occur outside a subject's body. The assays include cell-based assays that utilize live or dead cells, and may also include cell-free assays that do not utilize intact cells.

[0133]

[0147] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody. and includes human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab, and UCHT-1. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0134]

[0148] The term "OKT-3" (also referred to herein as "OKT3") refers to the mature T "CD3" refers to monoclonal antibodies, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of T cells, or biosimilars or variants thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or its variants, conservative amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are provided in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and has been assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and has been assigned catalog number 86022706.

[0135] [Table 1]

[0136]

[0149] The term "IL-2" (also referred to herein as "IL2") refers to interleukin-2 (IL-2). "IL-2" refers to the T cell growth factor known as IL-2, and includes all forms of IL-2, including its human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, 22 million I / O per single-use vial). (commercially available in the U.S. from multiple suppliers), as well as forms of recombinant IL-2 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 Other commercially available equivalents from vendors are included. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 4). The term IL-2 also includes the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA, and is used herein as a pegylated IL-2 prodrug. Also encompassed are pegylated forms of IL-2, as described in U.S. Patent Application Publication Nos. 2014 / 0328791 A1 and WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Other forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0137] [Table 2]

[0138]

[0150] The term "IL-4" (also referred to herein as "IL4") refers to interleukin-4 (IL-4). IL-4 refers to a cytokine known as IL-4, which is produced by Th2 T cells, as well as by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Activation by IL-4 subsequently leads to the differentiation of Th2 T cells. The cells produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is available from 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 CYT-212). It is commercially available from several suppliers, including Gibco (Log No. CTP0043). Use in the present invention The amino acid sequence of a suitable recombinant human IL-4 is provided in Table 2 (SEQ ID NO: 5).

[0139]

[0151] The term "IL-7" (also referred to herein as "IL7") refers to an interleukin-7 (IL7) It refers to a glycosylated tissue-derived cytokine known as ikine 7, which is available from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 shares a common receptor α IL-4 binds to the IL-7 receptor, a heterodimer consisting of a γ-chain receptor, which provides a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-4 suitable for use in the present invention is available from ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog). It is commercially available from several suppliers, including ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, 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 a recombinant human IL-7 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 6).

[0140]

[0152] The term "IL-15" (also referred to herein as "IL15") refers to an interferon-like protein (IL-15). IL-15 refers to the T cell growth factor known as IL-15, and includes all forms of IL-2, including its human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is available from 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, It is commercially available from several sources, including (catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 7).

[0141]

[0153] The term "IL-21" (also referred to herein as "IL21") refers to an interleukin-21 (IL-21) "IL-21" refers to the pleiotropic cytokine protein known as IL-21, and includes all forms of IL-21, including its human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated by reference. IL-21 is primarily a cytotoxic agent that stimulates natural killer T cells and activated human CD4 + Recombinant human IL-21 is produced by T cells. It is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is available from several 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 provided in Table 2 (SEQ ID NO: 8).

[0142]

[0154] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated The exact dosage of the compositions of the present invention can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, pharmaceutical compositions comprising the genetically modified cytotoxic lymphocytes described herein are administered in a dose of 100 mg / kg or more. 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 ,10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 , or 10 9 ~10 10 It can be stated that the genetically modified cytotoxic lymphocyte compositions may be administered at a dosage of 1000 mg / kg body weight (1000 mg / kg body weight) (including all integer values ​​within these ranges). The genetically modified cytotoxic lymphocyte compositions may also be administered multiple times at these dosages. The genetically modified cytotoxic lymphocytes may be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient will depend on the physician. This can be readily determined by one of ordinary skill in the art by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0143]

[0155] The term "hematological malignancies" includes, but is not limited to, tumors of the blood, bone marrow, lymph nodes, The term "B-cell hematological malignancies" refers to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including hematopoietic and lymphoid 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 myelocytic leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0144]

[0156] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, which may provide a supportive microenvironment within which the cancer cells are dispersed.

[0145]

[0157] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Examples include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs).

[0146]

[0158] The term "microenvironment" as used herein refers to the microenvironment of a solid tumor or a hematological tumor. The term "tumor microenvironment" as used herein may refer to the entire tumor microenvironment or individual cell subsets within the microenvironment. Tumor microenvironment, as used herein, is defined as described in Swartz, et al., Cancer Res., 2012, 72, 2473. As described in [the text], tumor clearance 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 tumors from host immunity, foster therapeutic resistance, and provide a niche for the development of overt metastases." Tumors express antigens that must be recognized by T cells, but tumor clearance by the immune system is rare due to the immunosuppressive microenvironment.

[0147]

[0159] In one embodiment, the present invention includes a method of treating cancer with a population of rTILs. , wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of the rTILs of the present invention. In some embodiments, the rTIL population may be provided with an eTIL population, wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of the rTILs and eTILs of the present invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for two days (days 27 and 26 prior to rTIL infusion) and fludarabine 25 mg / m2 / day for five days (days 27-23 prior to rTIL infusion). In one embodiment, after non-myeloablative chemotherapy and rTIL infusion of the present invention (day 0), the patient receives an intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours to a physiologically tolerated dose.

[0148]

[0160] Experimental findings suggest that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes may be beneficial for the treatment of It has been suggested that removal of nodal T cells and competing elements of the immune system ("cytokine sinks") plays an important role in enhancing therapeutic efficacy. Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") on patients prior to introducing the rTILs of the present invention.

[0149]

[0161] The terms "co-administration," "co-administer," "administered in conjunction with," and "concurrently with" As used herein, "administered in combination with," "simultaneously," and "concurrently" encompass administration of two or more active pharmaceutical ingredients (e.g., in preferred embodiments of the invention, at least one potassium channel agonist in combination with multiple TILs) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. This 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, with simultaneous administration in separate compositions and administration in a composition in which both agents are present being preferred.

[0150]

[0162] The term "effective amount" or "therapeutically effective amount" refers to any amount of a compound used in the treatment of a disease, including but not limited to the treatment of a disease. "Therapeutically effective" refers to an amount of a compound or combination of compounds as described herein sufficient to achieve the intended application. A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition under treatment (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. The term also applies to a dose that will produce a particular response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system that carries the compound.

[0151]

[0163] The terms "treatment," "treating," "treat," and the like refer to the desired pharmacological and "Treatment" refers to achieving a therapeutic and / or physiological effect. The effect may be prophylactic, meaning that a disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that a disease and / or adverse effects resulting from the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of 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., halting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a disease state, e.g., in the case of a vaccine.

[0152]

[0164] The term "heterologous" when used in reference to a portion of a nucleic acid or protein Indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a novel 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 comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0153]

[0165] The terms "sequence identity," "percent identity," and "sequence identity" in the context of two or more nucleic acids or polypeptides are used interchangeably. "Sequence identity," "sequence identity," and "sequence percent identity" (or their equivalents, e.g., "99% identity") refer to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned (introducing gaps as necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art that can be used to achieve alignment of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST program available from the U.S. government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR can be used to align sequences. Further publicly available software programs are available for use with the alignment software. Those skilled in the art can determine appropriate parameters to maximize alignment with a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0154]

[0166] As used herein, the term "variant" includes, but is not limited to, a variant of a reference antigen. The term "variant" encompasses antibodies or fusion proteins containing an amino acid sequence that differs from that of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to the antigen. The term "variant" also includes pegylated antibodies or proteins.

[0155]

[0167] The term "in vivo" refers to an event that takes place inside a subject's body.

[0156]

[0168] The term "in vitro" refers to events that occur outside a subject's body. The assays include cell-based assays that utilize live or dead cells, and may also include cell-free assays that do not utilize intact cells.

[0157]

[0169] The term "rapid expansion culture" refers to a culture that expands at least about three-fold (or four-fold, five-fold, or six-fold) over a one-week period. By rapid expansion is meant an increase in the number of antigen-specific TILs by at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a one week period, or most preferably by at least about 100-fold over a one week period. Several rapid expansion protocols are outlined below.

[0158] III. Re-stimulation of cryopreserved TILs

[0170] As discussed herein, the present invention provides a method for cryopreserving cells prior to transplantation into a patient. The present invention relates to restimulating TILs to increase their metabolic activity, and thus their relative health, and to methods for testing said metabolic health. Generally, as outlined herein, TILs are generally obtained from patient samples and manipulated to expand their numbers before transplantation into the patient. In some embodiments, the TILs may optionally be genetically manipulated as discussed below and then cryopreserved. After thawing, the TILs are then restimulated to increase their metabolism before infusion into the patient.

[0159]

[0171] The "step" names A, B, C, etc. below refer to Figure 11. The order of steps in 11 is exemplary, and any combination or order of steps, as well as additional steps, repetition of steps, and / or omission of steps, is contemplated by the present application and methods disclosed herein.

[0160] A. Step A: Obtain a patient tumor sample

[0172] Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then The TILs are expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

[0161]

[0173] Patient tumor samples are generally obtained by surgical extraction using methods known in the art. The tumor sample may be obtained by resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Generally, the tumor sample may be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be from a liquid tumor, such as a tumor obtained from a hematological malignancy. Solid tumors include, but are not limited to, breast cancer, The TILs may be of any cancer type, including pancreatic, prostate, colorectal, lung, brain, renal, gastric, and skin cancers (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, which have been reported to have particularly high levels of TILs.

[0162]

[0174] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, which may provide a supportive microenvironment within which the cancer cells are dispersed.

[0163]

[0175] The term "hematological malignancies" includes, but is not limited to, tumors of the blood, bone marrow, lymph nodes, The term "B-cell hematological malignancies" refers to cancers and tumors of hematopoietic and lymphoid tissues in mammals, including hematopoietic and lymphoid 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 myelocytic leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0164]

[0176] Once obtained, tumor samples are typically cut into sections of 1 to approximately 8 mm using sharp scraping. 3 fragmented into small pieces Approximately 2 to 3 mm 3are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests are prepared in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamic acid, 100 mM erythritol, 100 mM urea ...urea, 100 mM urea, 100 Tumor digests may be produced by incubation in a 100% soluble medium containing 100% TILs (100% TILs, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension at the end of this process contains numerous red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides may be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 A1 (the disclosure of which is incorporated herein by reference), may also be used. Any of the aforementioned methods may be used in any of the embodiments described herein for the methods of expanding TILs or treating cancer.

[0165]

[0177] In some embodiments, fragmentation can be physical, including, for example, peeling and digestion. In some embodiments, fragmentation includes fragmentation. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is exfoliation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.

[0166]

[0178] In some embodiments, if the tumor is a solid tumor, e.g., the tumor shown in FIG. As shown in Step A, after obtaining a tumor sample, the tumor undergoes physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented and each individual tumor is fragmented for the first expansion culture. Two, three, or four fragments or pieces are placed in each container. In some embodiments, the tumor is fragmented and three or four fragments or pieces are placed in each container for the first expansion culture. In some embodiments, the tumor is fragmented and four fragments or pieces are placed in each container for the first expansion culture.

[0167]

[0179] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 is.

[0168]

[0180] In some embodiments, the TILs are obtained from tumor digests. In this embodiment, tumor digests were generated by incubation in 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). Tumors were placed in enzyme medium. After the incubation, the tumor may be mechanically dissociated for about 1 minute. The solution may then be incubated at 37°C under 5% CO2 for 30 minutes, and then it may be mechanically disrupted again for about 1 minute. After again incubating at 37°C under 5% CO2 for 30 minutes, the tumor may be mechanically disrupted a third time for about 1 minute. In some embodiments, if large tissue debris was present after the third mechanical disruption, the sample was subjected to one or two additional rounds of mechanical dissociation, with or without an additional 30-minute incubation at 37°C under 5% CO2. In some embodiments, if the cell suspension at the end of the final incubation contained a large number of red blood cells or dead cells, density gradient separation using Ficoll may be performed to remove such cells.

[0169]

[0181] In some embodiments, the harvested cell suspension prior to the first expansion step The fluid is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0170]

[0182] In some embodiments, the cells are subjected to step B, which is described in more detail below. Before proceeding, the sample may optionally be frozen after collection and stored frozen.

[0171] B. Step B: First Expansion Culture

[0183] In some embodiments, the first expansion of TILs (first expansion or first The TILs obtained by this process may be optionally characterized for phenotypic characteristics and metabolic parameters as described herein. In some embodiments, the TILs are frozen (i.e., frozen after step D as shown in FIG. 11 ) after the first expansion (e.g., step B as shown in FIG. 11 ), followed by an additional second expansion (e.g., second step D as shown in FIG. 11 , which may include what may be referred to as a restimulation REP step) as described below and herein. In some embodiments, the TILs are frozen (i.e., frozen after step D as shown in FIG. 11 ) after the first expansion (e.g., step B as shown in FIG. 11 ). The cells are then stored (i.e., cryopreserved) until phenotyping for selection, and then thawed before proceeding to one or more second expansion steps (e.g., one or more expansion steps according to step D as shown in Figure 11).

[0172]

[0184] In some embodiments, after obtaining a tumor sample (e.g., as shown in FIG. 11), If the cells are frozen (such as during step A of the cage), the cells are thawed before the first expansion culture (eg, step B as shown in Figure 11).

[0173]

[0185] In an embodiment, TIL cultures are performed in 24-well plates, e.g., Costar24 Started using well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY) If used, each well contained 1 × 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6Tumor digest cells or 1 tumor fragment can be seeded. In some embodiments, tumor fragments are about 1 mm 3 ~10mm 3 is.

[0174]

[0186] After preparation of tumor fragments, the resulting cells (i.e., fragments) are more abundant than tumor and other cells. The primary cells are cultured in serum containing IL-2 under conditions favorable for TIL growth. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2 (or in some cases in the presence of an aAPC cell population, as outlined herein). This primary cell population is cultured for several days, generally 10-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion culture contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20-10 IU per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg of IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL IL-2 to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL IL-2 to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In certain embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3,000 IU / mL of IL-2. In some embodiments, the cell culture medium contains 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. Contains IL-2 between 0 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.

[0175]

[0187] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The cultures are grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10-40 x 10 cells in 10-40 mL of IL-2-containing CM. 6 10 live tumor digest cells or 5-30 tumor fragments were loaded. Each 4-well plate was incubated at 37°C under 5% CO in a humidified incubator. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onwards, half of the medium was replaced every 2-3 days. In some embodiments, the CM is CM1 as described in the Examples (see Example 5). In some embodiments, the first expansion culture is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

[0176]

[0188] In some embodiments, the first TIL expansion culture is performed on days 11, 12, 13, or 4. In some embodiments, the first TIL expansion culture may be continued for 11 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 12 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 13 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 14 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 15 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 16 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 17 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 18 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 19 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 20 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 21 days.

[0177] C. Step C: Transition from the first expansion culture to the second expansion culture

[0189] In some embodiments, from the first expansion culture (e.g., as shown in FIG. 11 ), The TILs obtained (from step B as provided in FIG. 11 ) are stored until phenotyping for selection. In some embodiments, the TILs obtained from the first expansion culture are cryopreserved after the first expansion culture and before the second expansion culture. In some embodiments, the TILs are cryopreserved as part of the transition from the first expansion culture to the second expansion culture. For example, in some embodiments, the TILs are cryopreserved after step B and before step D as shown in FIG. 11 . In some embodiments, the TILs are cryopreserved and thawed as part of the transition from the first expansion culture to the second expansion culture. For example, in some embodiments, the TILs are cryopreserved after step B and then thawed before proceeding to step D (as provided in FIG. 11 ). In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 22, 23, 24, 25, 26, 27, 28, 29, or 30 days from the time fragmentation occurred. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 22 to 30 days from the time fragmentation is performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 24 to 30 days from the time fragmentation is performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 25 to 30 days from the time fragmentation is performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 26 to 30 days from the time fragmentation was performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 28 to 30 days from the time fragmentation was performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 30 days from the time fragmentation was performed.

[0178] D. Step D: Second Expansion Culture

[0190] In some embodiments, a second expansion culture of TILs or a second TIL expansion culture The culture (which may include expansion, sometimes referred to as REP) can be performed using any TIL flask or vessel known to one of skill in the art. In some embodiments, the second TIL expansion culture may continue for 14, 15, 16, 17, 18, 19, 20, 21, or 22 days. In some embodiments, the second TIL expansion culture may continue for about 14 days to about 22 days. In some embodiments, the second TIL expansion culture may continue for about 14 days to about 20 days. In some embodiments, the second TIL expansion culture may continue for about 14 days to about 18 days. In some embodiments, the second TIL expansion culture may continue for about 14 days to about 16 days. In some embodiments, the second TIL expansion culture may continue for about 14 days.

[0179]

[0191] In some embodiments, the second expansion culture is performed in supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells).

[0180]

[0192] In some embodiments, a second expansion culture of TILs (called REP) is performed. The cells were cultured in T-175 flasks and gas-permeable bags (Tran KQ, Zhou J, Durflinger KH, et al., 2008, J Immunother., 31:742-751 and Dudley ME, Wunderlich JR, Shelton TE, et al., 2003, J Immunother., 26:332-342) or gas-permeable bags (Tran KQ, Zhou J, Durflinger KH, et al., 2008, J Immunother., 31:742-751 and Dudley ME, Wunderlich JR, Shelton TE, et al., 2003, J Immunother., 26:332-342) as previously described. In some embodiments, the second culture may be performed using a permeable G-Rex flask. In some embodiments, the second expansion is performed using a gas-permeable G-Rex flask. For the second expansion culture, approximately 1 x 10 6 TILs are suspended in approximately 150 mL of medium and added to each T-175 flask. TILs are cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells are cultured in a 1:100 ratio of CM supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3 and AIM-V medium. The T-175 flasks are incubated at 37°C under 5% CO2. In some embodiments, 5 days into the second expansion culture, half of the medium is replaced with 50 / 50 medium containing 3000 IU / mL of IL-2. In some embodiments, on day 7, cells from two T-175 flasks are combined into a 3L bag, and 300 mL of TIL suspension is added to the bag with 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2. The cells in each bag can be counted daily or every two days. Add fresh medium and culture the cells to a density of approximately 0.5 to 2.0 x 10 6 cells / mL.

[0181]

[0193] In some embodiments, a second expansion culture of TILs (called REP) is performed. (which may include expansion cultures) is 100 cm 2 It can be performed in a 500 mL gas-permeable flask with a gas-permeable silicone bottom (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) (Figure 1), and has a concentration of 3000 IU / mL. Approximately 5 × 10 cells were cultured in 400 mL of 50 / 50 medium supplemented with 100 mg of IL-2 and 30 ng / mL of anti-CD3 (OKT3). 6 or 10 x 10 6 TILs are cultured with irradiated allogeneic PBMCs at a ratio of 1:100. The G-Rex100 flasks can be incubated at 37°C under 5% CO2. In some embodiments, 5 days into the second expansion culture, 250 mL of supernatant is removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TILs are then cultured in a 5% human A solution. The TIL pellet may be resuspended in 150 mL of fresh medium containing AB serum and 3000 IU / mL of IL-2 and added back to the original G-Rex100 flask. In embodiments where TILs are serially expanded in G-Rex100 flasks, on day 7, the TILs in each G-Rex100 are suspended in the 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots that may be used to seed three G-Rex100 flasks. Each flask may then receive 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2. The G-Rex100 flasks are incubated at 37°C under 5% CO2 until they enter the second expansion culture, at which point the TILs reach the 300 mL of medium present in each flask. The cell suspension may then be divided into three 100 mL aliquots that may be used to seed three G-Rex100 flasks. Each flask may then receive 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2. The G-Rex100 flasks are incubated at 37°C under 5% CO2 until they reach the 400 mL mark, at which point the TILs reach the 400 mL mark and enter the second expansion culture. After 2 days, each G-Rex100 flask was inoculated with 150 mL of AIM-V containing 3000 IU / mL of IL-2. In some embodiments, the cells are harvested on day 14 of culture.

[0182]

[0194] In some embodiments, a second expansion culture of TILs (called REP) is performed. Expansion (which may include expansion of TILs in a gas-permeable container) can be performed in a gas-permeable container. 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). In one embodiment, expansion of TIL numbers is performed at approximately 1 x 10 9 ~Approx. 1×10 11 Antigen-presenting feeder cells are used. Non-specific T cell receptor stimulation can include, for example, about 30 ng / ml OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). TILs can be generated by any Further stimulation of TILs in vitro with one or more antigens of the cancer, including one or more epitopes thereof, optionally expressed from a vector, such as human leukocyte antigen A2 (HLA-A2)-binding peptides, e.g., 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M), can be rapidly expanded in the presence of a T cell growth factor, optionally 300 IU / mL IL-2 or IL-15. Other suitable antigens include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same one or more antigens of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0183]

[0195] In some embodiments, a second expansion culture of TILs (called REP) is performed. Expansion culture (which may include the expansion of the cells) can be performed in a 100 cm gas-permeable silicone-bottomed, 500 mL capacity gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) containing 5% human AB serum, 3000 mL of PBS. 5 × 10 cells in 400 mL of 50 / 50 medium supplemented with 1 U / mL IL-2 and 30 ng / mL anti-CD3 (OKT3). 6 or 10 x 10 6 TILs can be cultured with aAPCs at a ratio of 1:100. G-Rex 100 flasks can be incubated at 37°C under 5% CO2 for 5 days. After this, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU / mL IL-2 and added back to the original G-Rex 100 flask. When serially expanding TILs in G-Rex 100 flasks, the TILs of each G-Rex 100 flask can be resuspended on day 7. The L may be suspended in the 300 mL of medium present in each flask, and the cell suspension may be divided into three 100 mL aliquots, which are used to seed three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 can be added to each flask. The G-Rex 100 flasks can be incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 can be added to each G-Rex 100 flask. Cells can be harvested on day 14 of culture.

[0184]

[0196] In one embodiment, the second expansion culture (including the expansion culture referred to as REP) Bulk TILs were cultured in 150 ml of medium containing 100-fold or 200-fold excess inactivated feeders. The cells are mixed with 30 mg / mL OKT3 anti-CD3 antibody and 3000 IU / mL IL-2 in flasks. Media replenishment is performed (typically two-thirds media replenishment via respiration with fresh media) until the cells are transferred to separate growth chambers. Separate growth chambers include GRex flasks and gas-permeable vessels, as discussed more fully below.

[0185]

[0197] In another embodiment, a second expansion culture (including an expansion culture called REP) ) is performed, which further includes selecting TILs for superior tumor response. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058 A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs for superior tumor response.

[0186]

[0198] Optionally, a second expansion culture (including an expansion culture referred to as a REP expansion culture) Following this, cell viability assays can be performed using standard assays known in the art. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows assessment of viability. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to the Cellometer K2 Image Cytometer automated cell counter protocol described, for example, in Example 2.

[0187]

[0199] In some embodiments, the cells are split into two or more vessels or flasks. Grow for a total time of 7, 8, 9, 10, or 11 days before the second expansion culture.

[0188]

[0200] In some embodiments, the second expansion culture medium (e.g., CM2 or a second The cell culture medium (sometimes referred to as cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

[0189]

[0201] 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 some embodiments, 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 some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is 1:50 to 1:300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is 1:100 to 1:200.

[0190]

[0202] In one embodiment, the TIL expansion procedures described herein include: Excessive amounts of feeder cells are required during the second expansion culture (including, for example, the expansion culture referred to as REP TIL expansion culture). In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used instead of PBMCs.

[0191]

[0203] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment, and R Used in EP procedures.

[0192]

[0204] In some embodiments, the growth medium in the first expansion culture comprises IL-2 or In some embodiments, the IL is recombinant human IL-2 (rhIL-2 In some embodiments, the IL-2 stock solution contains 20-30 x 10 per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20-10 IU per 1 mg vial. 6IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg of IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL IL-2 to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL IL-2 to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium comprises about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In certain embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3,000 IU / mL of IL-2. In certain embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In certain embodiments, the cell culture medium comprises between 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.

[0193]

[0205] In some embodiments, the cell culture medium of the second expansion culture also contains anti-CD3 The cell culture medium also contains an antibody. In some embodiments, the cell culture medium comprises an OKT3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT3 antibody. In certain embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 μg / mL of the OKT3 antibody. In certain embodiments, the cell culture medium contains 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 of OKT3 antibody.

[0194]

[0206] In some embodiments, the anti-CD3 antibody in combination with IL-2 inhibits TIL recruitment. This effect can be seen with full-length antibodies as well as Fab and F(ab')2 fragments, the former being generally preferred; see, e.g., Tsoukas et al. al., J. Immunol. 1985, 135, 1719 (hereby incorporated by reference in its entirety). As one of skill in the art will appreciate, the various methods that find use in the present invention include: There are a number of suitable anti-human CD3 antibodies, including anti-human CD3 polyclonal and monoclonal antibodies from a variety of mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In a particular embodiment, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). (It is being done).

[0195]

[0207] In some embodiments, the cells in the second expansion culture are cultured in a manner known in the art. As is known in the art, the cells are grown in culture medium containing high doses of cytokines, particularly IL-2.

[0196]

[0208] Alternatively, a combination of cytokines can be used for the second expansion of TILs. and combinations of two or more of IL-2, IL-15, and IL-21 are further possible, as generally outlined in WO 2015 / 189356 and 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, IL-21 and IL-2, and IL-15 and IL-21, the latter finding particular use in many embodiments. As described therein, the use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells.

[0197] E. Optional Step D: Repeat Second Expansion Culture

[0209] In some embodiments, the second expansion culture is performed one or more times, i.e., the second The expansion is repeated. For example, in some embodiments, the second expansion of step D as shown in FIG. 11 is repeated one or more times. In some embodiments, the second expansion is referred to as an additional second expansion. In some embodiments, where the second expansion is performed more than once (i.e., the second expansion is repeated), this may include a procedure referred to as a TIL rapid expansion protocol. In some embodiments, the TIL cell population is expanded in number after harvesting and the first expansion. This process is generally referred to in the art as a rapid expansion process (REP), and may be repeated. The second expansion culture performed may include an expansion culture referred to as reREP. This entire protocol can generally be accomplished using culture medium in a gas-permeable container containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody. In some embodiments, one or more subsequent second expansion cultures are performed as described above. In some embodiments, one or more second expansion cultures are performed as provided under step D of FIG. 11 and before step E as provided in FIG. 11. In some embodiments, one, two, three, four, or more second expansion cultures are performed as described above. In some embodiments, one, two, three, four, or more second expansion cultures are performed as provided in step D of FIG. 11 before step E of FIG. 11. In some embodiments, two second expansion cultures are performed as described above. In some embodiments, two second expansion cultures are performed as provided in step D of FIG. 11 before step E of FIG. 11. In some embodiments, three second expansion cultures are performed as described above. In some embodiments, three rounds of second expansion culture are performed as provided in step D of Figure 11 before step E of Figure 11. In some embodiments, four rounds of second expansion culture are performed as described above. In some embodiments, four rounds of second expansion culture are performed as provided in step D of Figure 11 before step E of Figure 11.

[0198]

[0210] In some embodiments, a second expansion culture of TILs (e.g., step 11 of FIG. 11) is performed. A repeat of the restimulation step, i.e., a second expansion cycle (e.g., as shown in Figure 1), can be referred to as restimulation of the TILs. In some embodiments, the repeated second expansion (which may include expansion referred to as a restimulation step ("reREP")) is performed on cells that have been cryopreserved. In some embodiments, the TILs are cryopreserved after step D. In some embodiments, after the initial second expansion in step D, the cells may be cultured in regular medium, e.g., "quiescent" medium, and then one or more second expansion steps are performed. In some embodiments, the quiescent medium includes IL-2. In some embodiments, the quiescent medium does not include IL-2. In some embodiments, the quiescent medium is a standard cell culture medium known in the art. In some embodiments, the quiescent medium is a medium such as AIM-V, DMEM, or other commercially available media. In some embodiments, the quiescent medium is AIM-V, DMEM / F12, MEM, RPMI, OptiMEM, IMDM, or any other standard medium known in the art. is.

[0199]

[0211] Generally, as discussed herein, TILs are initially The TILs are prepared by obtaining a primary TIL population from a tumor resected from a patient as described above (the "primary cell population" or "first cell population"). This is followed by an initial bulk expansion culture utilizing culturing the cells with IL-2 to form a second cell population (sometimes referred to herein as the "bulk TIL population" or "second population"). In some embodiments, this is also referred to as the initial or first expansion culture.

[0200]

[0212] Next, the bulk TIL population (e.g., the TILs obtained from step A of Figure 11) is cultured. The expanded TIL population (e.g., a population obtained from the first expansion, e.g., a first expansion as described in step B of FIG. 11 ) is subjected to a REP step, sometimes referred to as a first expansion, in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (APCs), where the APCs generally comprise peripheral blood mononuclear cells (PBMCs; or, alternatively, antigen-presenting cells are used, as discussed herein), where the rapid expansion (e.g., a second expansion as provided in step D of FIG. 11 ) is performed for at least 14 days. As discussed herein, the medium may also contain a combination of IL-2, IL-15, and / or IL-23, rather than IL-2 alone. In some embodiments, the expanded TIL population after this second expansion (e.g., after step D of FIG. 11 ) is at least 50-fold or 100-fold more numerous than the second TIL population (e.g., the TIL population obtained from step B of FIG. 11 ). In some embodiments, the TIL population obtained after the second expansion in step D of Figure 11 is 50-fold or 100-fold more numerous than the TILs obtained from the first expansion in step B of Figure 11. TILs are measured by cell counting methods known in the art, including those described in the Examples provided herewith, including Examples 1, 2, and 3. In some embodiments, TILs are counted using a K2 cell counter. In some embodiments, TILs are counted using a Cellometer IC2 Image cytometer.

[0201]

[0213] In some embodiments, after the second expansion, as discussed herein, The obtained TIL population (sometimes referred to as a third TIL population or REP cell population) is removed from the supplemented cell culture medium (e.g., the culture medium used in step D of Figure 11 or the medium referred to as CM2 in the examples) and cryopreserved in a storage medium (e.g., medium containing 5% DMSO) optionally before performing an additional second expansion culture step.

[0202]

[0214] Optionally, the TILs are cultured after the second expansion and after an additional second expansion. In some embodiments, the TILs are cryopreserved after performing step D of FIG. 11 and before performing the additional step D of FIG. 11. In some embodiments, the cryopreserved TILs are thawed before performing the additional second expansion culture. In some embodiments, the cryopreserved TILs are thawed before performing the additional step D as provided in FIG. 11. In some embodiments, the TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture medium + 5% DMSO. Alternatively, the cells are removed from the supplemented cell culture medium (e.g., the culture medium used in step D of FIG. 11) and cultured in a quiescent medium. Such media include those described in Examples 1 and 5 and other Examples provided herewith. In some embodiments, the quiescent medium may comprise a medium containing IL-2. In some embodiments, the quiescent medium may be the medium referred to in the Examples as CM1.

[0203]

[0215] Additional secondary expansion cultures (including expansion cultures designated reREP) were prepared by thawing The additional second expansion is performed on either the cultured or resting cells using supplemented cell culture medium (e.g., medium as provided in step D of Figure 11) containing IL-2, OKT-3, and feeder cells (e.g., antigen-presenting cells), generally comprising peripheral blood mononuclear cells (PBMCs; or, alternatively, using antigen-presenting cells as discussed herein), where the additional second expansion is performed for at least 14 days. As discussed herein, the medium may also contain a combination of IL-2, IL-15, and / or IL-23, rather than IL-2 alone.

[0204]

[0216] This allows these expanded cultured TILs to become a second TIL population (e.g., bulk TILs). A population of expanded TILs is obtained that is characterized by exhibiting increased effector T cell and / or central memory T cell subpopulations relative to the starting TILs. In some embodiments, these expanded TILs are the TILs obtained from step D of FIG. 11.

[0205]

[0217] In some embodiments, memory T cells are cells (constitutively expressing CCR7 and CD62L). See Sallusto, et al., Annu. Rev. Immunol., 2004, 22:745-763. , which is incorporated herein by reference in its entirety.

[0206]

[0218] Thus, the present invention provides post-thawing benefits that result in increased metabolic health, such as glycolysis and respiration. In some embodiments, the method includes providing a population of thawed cryopreserved TILs, which are then treated to increase their metabolic health to achieve optimal therapy upon infusion into a patient.

[0207] F. Step E: Harvest TILs from Step D

[0219] After the second expansion step, the cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four, or more second expansion steps. In some embodiments, the TILs are harvested after one, two, three, four, or more second expansion steps according to step D as provided in FIG.

[0208]

[0220] The TILs may be collected by any suitable and sterile method, including, for example, by centrifugation. Methods for recovering TILs are well known in the art, and any such known method can be used with the present process.

[0209] G. Step F: Final Formulation and / or Transfer to Infusion Bag

[0221] As provided in an exemplary sequence in FIG. 11 and as described above and in detail herein. After steps A-E are completed as outlined, the cells are transferred to a container for use in administration to a patient. In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, the TILs are transferred to a container for use in administration to a patient.

[0210]

[0222] In one embodiment, the TILs expanded using the APCs of the present disclosure: They are administered to patients as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0211] 1. Pharmaceutical Compositions, Dosages, and Administration Regimen

[0223] In one embodiment, the TILs expanded using the APCs of the present disclosure: They are administered to patients as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting about 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0212]

[0224] Any suitable dose of TILs can be administered. Therefore, a suitable dosage requires a therapeutically sufficient number of TILs. In some embodiments, about 2.3 x 10 10 ~Approx. 13.7×10 10 TILs were administered, with an average of approximately 7.8 x 10 10In one embodiment, about 1.2 x 10 TILs. 10 ~Approx. 4.3×10 10 In some embodiments, about 3 x 10 TILs are administered. 10 ~Approx. 12×10 10 In some embodiments, about 4 x 10 TILs are administered. 10 ~About 10×10 10 In some embodiments, about 5 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, about 6 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, about 7 x 10 TILs are administered. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dosage is about 2.3 x 10 TILs. 10 ~Approx. 13.7×10 10 In some embodiments, a therapeutically effective dosage is about 7.8 x 10, particularly when the cancer is melanoma. 10 In some embodiments, the therapeutically effective dosage is about 1.2 x 10 TILs. 10 ~Approx. 4.3×10 10 In some embodiments, the therapeutically effective dosage is about 3 x 10 TILs. 10 ~Approx. 12×10 10 In some embodiments, the therapeutically effective dosage is about 4 x 10 TILs. 10 ~About 10×10 10 In some embodiments, the therapeutically effective dosage is about 5 x 10 TILs. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dosage is about 6 x 10 TILs. 10 ~Approx. 8×10 10 In some embodiments, the therapeutically effective dosage is about 7 x 10 TILs. 10 ~Approx. 8×10 10 This is the individual TIL.

[0213]

[0225] In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is , about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11, 9×10 11 , 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 It is an individual. In the form, the number of TILs provided in the pharmaceutical composition of the present invention is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , and 5 × 10 12 ~1×10 13 In some embodiments, the therapeutically effective dosage is about 1 x 10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×106 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×10 11 、3×10 11 、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12, 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 There are individuals.

[0214]

[0226] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.0% of the pharmaceutical composition. less than 4%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v.

[0215]

[0227] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention represents 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% of the pharmaceutical composition 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or greater than 0.0001% w / w, w / v, or v / v.

[0216]

[0228] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.0 The range is 9% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w / w, w / v, or v / v.

[0217]

[0229] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, or about 0.1% to about 0.9% w / w, w / v, or v / v of the pharmaceutical composition.

[0218]

[0230] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is , 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0. 95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.25g, 0.2g, 0.15g, 0.1g, 0 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

[0219]

[0231] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is , 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008 g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.0025g, 0.003g, 0.0035g, 0.004g, 0 .0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.00 85g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g , 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0.08g, 0.085g, 0.0 9g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g, 0.5g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or more than 10 g.

[0220]

[0232] The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend on the route of administration, the dosage form of the compound, the sex and age of the subject, the body weight of the subject, and the preferred choice and experience of the attending physician. Clinically established dosages of TILs may also be used where appropriate. The amount of the pharmaceutical composition administered using the methods herein, including the dosage of TILs, will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the nature of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0221]

[0233] In some embodiments, the TILs may be administered in a single dose. Administration may be by injection, for example, intravenous injection. In some embodiments, the TILs may be administered in multiple doses. Administration may be once, twice, three times, four times, five times, six times, or more than six times per year. Administration may be monthly, once every two weeks, once a week, or once every other day. Administration of the TILs may be continued as long as necessary.

[0222]

[0234] In some embodiments, the effective dosage of TILs is about 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10, 8×10 10 , 9×10 10 , 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , and 9×10 13 In some embodiments, the effective dosage of TILs is 1 x 10 6 ~5×10 6 , 5×10 6 ~1×10 7 , 1×10 7 ~5×10 7 , 5×10 7 ~1×10 8 , 1×10 8 ~5×10 8 , 5×10 8 ~1×10 9 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 1×10 10 ~5×10 10 , 5×10 10 ~1×10 11 , 5×10 11 ~1×10 12 , 1×10 12 ~5×10 12 , and 5 × 10 12~1×10 13 It is a range of individuals.

[0223]

[0235] In some embodiments, the effective dosage of TILs is about 0.01 mg / kg to Approximately 4.3 mg / kg, approximately 0.15 mg / kg to approximately 3.6 mg / kg, approximately 0.3 mg / kg to approximately 3.2 mg / kg, approximately 0.35 mg / kg to approximately 2.85 mg / kg, approximately 0.15 mg / kg to approximately 2.85 mg / kg, approximately 0.3 mg to approximately 2.15 mg / kg, approximately 0.45 mg / kg to approximately 1.7 mg / k g, about 0.15 mg / kg to about 1.3 mg / kg, about 0.3 mg / kg to about 1.15 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.55 mg / kg to about 0.85 mg / kg, about 0.65 mg / kg to about 0.8 mg / kg, about 0.7 mg / kg to about 0.75 mg / kg, about 0.7 mg / kg to about 2.15 mg / kg, about 0.85 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.85 mg / kg, about 1.15 mg / kg to about 1.7 mg / kg, about 1.3 mg / kg to about 1.6 mg / kg, about 1.35 mg / kg to about 1.5 mg / kg, about 2.15 mg / kg to about 3.6 mg / kg, about 2.3 mg / kg to about 3.4 mg / kg, about 2.4 mg / kg to about 3.3 mg / kg, about 2.6 mg / kg to about 3.15 mg / kg, about 2.7 mg / kg to about 3 mg / kg, about 2.8 mg / kg to about 3 mg / kg, or about 2.85 mg / kg to about 2.95 mg / kg.

[0224]

[0236] In some embodiments, an effective dosage of TILs is from about 1 mg to about 500 mg. , about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 1 5mg to about 35mg, about 20mg to about 30mg, about 23mg to about 28mg, about 50mg to about 150mg, about 60mg to about 140mg, about 70mg to about 130mg, about 80m The range is from about 100 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, from about 98 mg to about 102 mg, from about 150 mg to about 250 mg, from about 160 mg to about 240 mg, from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, from about 195 mg to about 205 mg, or from about 198 mg to about 207 mg.

[0225]

[0237] Effective doses of TILs can be administered by intranasal and transdermal routes, intra-arterial injection, intravenous, They may be administered in single or multiple doses by any of the commonly accepted modes of administration for agents having similar utilities, including intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by implant, or by inhalation.

[0226] H. Optional Cell Viability Analysis

[0238] Optionally, after the first expansion culture of step B, a method known in the art for Cell viability assays can be performed using known standard assays. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows assessment of viability. Other assays used to test viability include, but are not limited to, the Alamar Blue assay; and the MTT assay.

[0227] 1. Cell Count, Viability, and Flow Cytometry

[0239] In some embodiments, cell number and / or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56, and any others disclosed or described herein, can be measured by antibody-based flow cytometry, such as, but not limited to, those commercially available from BD Biosciences (BD Biosciences, San Jose, CA) using a FACSCanto™ flow cytometer (BD Biosciences). Cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art, including, but not limited to, trypan blue staining.

[0228]

[0240] In some cases, bulk TIL populations are directly cultured using the protocols discussed below. The bulk TIL population can be immediately cryopreserved. Alternatively, the bulk TIL population can be subjected to REP, as discussed below, and then cryopreserved. Similarly, if the genetically modified TILs are to be used in therapy, the bulk or REP TIL population can be subjected to the appropriate therapeutic genetic modification.

[0229] 2.Cell culture

[0241] In one embodiment, the method for expanding TILs involves culturing a culture medium containing about 5,000 mL to about 2 This may include using 5,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, one or less types of cell culture medium are used to expand the number of TILs. Any suitable cell culture medium may be used, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, 50 μM ethanol ... and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this regard, the methods of the present invention advantageously reduce the amount of medium and number of media types required to expand the number of TILs. In certain embodiments, expanding the number of TILs can involve adding fresh cell culture medium to the cells (also referred to as feeding the cells) no more frequently than every two or three days. Expanding the number of cells in a gas-permeable container simplifies the procedures required to expand the number of cells by reducing the feeding frequency required for cell expansion.

[0230]

[0242] In some embodiments, the cell culture medium in the first and / or second gas permeable container The cell culture medium in the first and / or second gas-permeable containers is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and / or second gas-permeable containers does not contain β-mercaptoethanol (BME).

[0231]

[0243] In one embodiment, obtaining a tumor tissue sample from the mammal; cell culture medium obtaining TILs from the tumor tissue sample; and culturing a second gas-permeable container containing cell culture medium using aAPCs. The duration of the method includes expanding the number of TILs within about 14 to about 42 days, e.g., about 28 days.

[0232]

[0244] In one embodiment, the TILs are expanded in a gas-permeable container. Permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717 A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are expanded in gas-permeable bags. In one embodiment, TILs are expanded in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). The TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags. In one embodiment, the TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system comprises a gas-permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L. In one embodiment, the TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such an embodiment expands the cell population to about 5 x 10 5 cells / cm 2 From 10 x 10 6 ~30×10 6 cells / cm 2In some embodiments, this expansion is done without adding fresh cell culture medium to the cells (also referred to as feeding the cells). In some embodiments, this is without feeding, as long as there is about 10 cm of medium in the GRex flask. In some embodiments, this is without feeding, but one or more cytokines are added. In some embodiments, the cytokines can be added as a bolus without any need to mix the cytokine with the medium. Such vessels, devices, and methods are known in the art and have been used for the expansion of TILs, including, for example, U.S. Patent Application Publication No. 2014 / 0377739 A1, WO 2014 / 210036 A1, U.S. Patent Application Publication No. 2013 / 0115617 A1, WO 2013 / 188427 A1, U.S. Patent Application Publication No. 2011 / 0136228 A1, U.S. Patent No. 8,809,050 B2, WO 2011 / 072088 A2, U.S. Patent Application Publication No. 2016 / 0208216 A1, U.S. Patent Application Publication No. 2012 / 0244133 A1, WO 2012 / 129201 A1, U.S. Patent Application Publication No. 2013 / 0102075 A1, U.S. Patent No. 8,956,860 B2, and the like. B2, WO 2013 / 173835 A1, and U.S. Patent Application Publication No. 2015 / 0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292. Optional TIL Gene Modification

[0233]

[0245] In some embodiments, the TILs are optionally, but not limited to, high affinity TILs. They are genetically engineered to contain additional functionality, including T cell receptors (TCRs), for example, TCRs that target tumor-associated antigens such as MAGE-1, HER2, or NY-ESO-1, or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19).

[0234] I. Optional TIL cryopreservation

[0246] As discussed above in steps A-E, the entire TIL expansion process Cryopreservation can occur at various times throughout the body. In some embodiments, the bulk TIL population after the first expansion according to step B or the expanded TIL population after one or more second expansions according to step D may be cryopreserved. Cryopreservation generally involves freezing the TILs in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). This can be achieved by placing a population of TILs in a cryogenic vial. The cells in this solution are placed in a cryogenic vial, stored at -80°C for 24 hours, and optionally transferred to a nitrogen gas freezer for cryopreservation. See Sadeghi, et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, the TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture medium + 5% DMSO. In some embodiments, the TILs are cryopreserved according to the methods provided in Examples 8 and 9.

[0235]

[0247] If appropriate, remove the cells from the freezer and place them in a 37°C water bath for approximately 5 minutes. Thaw until a quarter of the cells are thawed. The cells are generally resuspended in complete medium and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as known in the art.

[0236] J. Phenotypic characterization of expanded TILs

[0248] In some embodiments, the TILs are expanded and then cultured to produce the desired TILs. The TILs are analyzed for expression of a number of phenotypic markers, including those listed above. In certain embodiments, expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the first expansion in step B. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transfer in step C. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transfer in step C and after cryopreservation. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the second expansion in step D. In some embodiments, the phenotypic characteristics of the TILs are analyzed after two or more expansions in step D. In some embodiments, the markers are selected from the group consisting of TCRab, CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the markers are selected from the group consisting of TCRab, CD57, CD28, CD4, CD27, CD56, and CD8a. In some embodiments, the markers are selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 markers is examined. In some embodiments, expression from one or more markers from each group is examined. In some embodiments, one or more of HLA-DR, CD38, and CD69 expression is maintained (i.e., does not exhibit a statistically significant difference) in fresh TILs compared to thawed TILs. In some embodiments, the activation state of TILs is maintained in thawed TILs.

[0237]

[0249] In certain embodiments, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatory marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, regulatory molecule expression is decreased in thawed TILs compared to fresh TILs. In some embodiments, expression of regulatory molecules LAG-3 and TIM-3 is decreased in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in CD4, CD8, NK, or TCRαβ expression. In some embodiments, there are no significant differences in CD4, CD8, NK, TCRαβ expression, and / or memory markers in fresh TILs compared to thawed TILs.

[0238]

[0250] In some embodiments, the memory markers are selected from CCR7 and CD62L. is selected from the group consisting of:

[0239]

[0251] In some embodiments, the viability of fresh TILs compared to thawed TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, the viability of both fresh and thawed TILs is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98%. In some embodiments, the viability of both fresh and thawed products is greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, or greater than 90%. In some embodiments, the viability of both fresh and thawed products is greater than 86%.

[0240]

[0252] In one embodiment, restimulated TILs are also assayed using cytokine release assays. In some embodiments, TILs can be assessed for interferon-7 (IFN-7) secretion in response to stimulation with either OKT3 or co-culture with autologous tumor digest. For example, in embodiments using OKT3 stimulation, TILs are washed extensively and plated at 1 x 10 in 0.2 mL CM onto 96-well flat-bottom plates pre-coated with 0.1 or 1.0 μg / mL OKT3 diluted in phosphate-buffered saline. 5 Duplicate wells are prepared with cells. After overnight incubation, the supernatant is collected and IFN-7 in the supernatant is measured by ELISA (Pierce / Endogen, Woburn, MA). For co-culture assays, 1 x 10 cells are used. 5 TIL cells are plated in 96-well plates together with autologous tumor cells (1:1 ratio). After 24 hours of incubation, supernatants can be collected and IFN-7 release can be quantified, for example, by ELISA.

[0241]

[0253] Flow cytometry analysis of cell surface biomarkers: TIL samples were aliquoted for flow cytometry analysis (see, e.g., Examples 7, 8, and 9).

[0242]

[0254] In some embodiments, the TILs are assessed for various regulatory markers. In some embodiments, the regulatory marker is selected from the group consisting of TCRα / β, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R, CCR7, CD62L, KLRG1, and CD122. In some embodiments, the regulatory marker is TCRα / β. In some embodiments, the regulatory marker is CD56. In some embodiments, the regulatory marker is CD27. In some embodiments, the regulatory marker is CD28. In some embodiments, the regulatory marker is CD57. In some embodiments, the regulatory marker is CD45RA. In some embodiments, the regulatory marker is CD45RO. In some embodiments, the regulatory marker is CD25. In some embodiments, the regulatory marker is CD127. In some embodiments, the regulatory marker is CD95. In some embodiments, the regulatory marker is IL-2R. In some embodiments, the regulatory marker is CCR7. In some embodiments, the regulatory marker is CD62L. In some embodiments, the regulatory marker is KLRG1. In some embodiments, the regulatory marker is CD122.

[0243] K. Metabolic health of expanded TILs

[0255] Restimulated TILs were either freshly harvested and / or thawed TILs. It is also characterized by a significant increase in basal glycolysis when compared with the normal period.

[0244]

[0256] TILs expanded with aEM3 aAPCs were compared with PBMC feeders. By comparison, spare respiratory capacity (SRC) and glycolytic reserve can be determined. The cellular mitochondria stress test is a method for directly measuring cellular oxygen consumption rate (OCR) using respiratory regulators that target components of the mitochondrial electron transport chain. Mitochondrial function is measured. Test compounds (oligomycin, FCCP, and a mixture of rotenone and antimycin A, described below) are continuously injected to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. These parameters and basal respiration are then used to calculate proton leak and spare respiratory capacity. Each regulator targets a specific component of the electron transport chain. Oligomycin inhibits ATP synthase (complex V), and the decrease in OCR after oligomycin injection correlates with mitochondrial respiration, which is associated with cellular ATP production. Carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) is an uncoupler that disrupts the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the electron transport chain is uninhibited, allowing maximal oxygen consumption by complex IV. FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal and basal respiration. Spare respiratory capacity (SRC) is a measure of a cell's ability to respond to increased energy demands. The third injection is a mixture of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination blocks mitochondrial respiration and allows for the calculation of non-mitochondrial respiration, which is driven by processes outside the mitochondria.

[0245]

[0257] In some embodiments, the metabolic assay is basal respiration. The expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 11 , including TILs referred to as reREP TILs) have a basal respiration rate that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is about 80% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is about 90% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the basal respiration rate is about 95% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the second expansion culture or second additional expansion culture TILs (e.g., including TILs designated reREP TILs, such as those described in step D of FIG. 11 ) have a basal respiration rate that is not statistically significantly different from the basal respiration rate of freshly harvested TILs.

[0246]

[0258] Generally, the second expanded TILs or additional second expanded TILs, e.g. TILs, such as those in step D (e.g., including TILs that have undergone an additional second expansion, designated reREP), have a spare respiratory capacity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is about 80% to about 99% of the basal respiratory rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is about 90% to about 99% of the basal respiratory rate of freshly harvested TILs. In some embodiments, the spare respiratory capacity is about 95% to about 99% of the basal respiratory rate of freshly harvested TILs. In some embodiments, the second expanded TILs or second additional expanded TILs (e.g., those described in step D of Figure 11, including TILs designated reREP TILs) have a spare respiratory capacity that is not statistically significantly different from the basal respiration rate of freshly harvested TILs.

[0247]

[0259] Generally, a second expanded TIL or a second additional expanded TIL (e.g. , including TILs referred to as reREP TILs, such as those described in step D of FIG. 11 , have a spare respiratory capacity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the metabolic assay measured is glycolytic reserve. In some embodiments, the metabolic assay is glycolytic reserve. In some embodiments, the metabolic assay is spare respiratory capacity. To measure cellular (respiratory) metabolism, cells were treated with inhibitors of mitochondrial respiration and glycolysis to determine a metabolic profile of the TILs consisting of the following measures: baseline oxidative phosphorylation (as measured by OCR), spare respiratory capacity, baseline glycolytic activity (as measured by ECAR), and glycolytic reserve. Metabolic profiles were measured using the Seahorse Combination Mitochondrial / Glycolysis Stress Test Assay (a commercially available kit from Agilent®), which is capable of determining the ability of cells to perform glycolysis upon the blockage of mitochondrial ATP production. In some embodiments, the cells are glucose starved and then infused with glucose, followed by the stress agent. In some embodiments, the stress agent is selected from the group consisting of oligomycin, FCCP, rotenone, antimycin A, and / or 2-deoxyglucose (2-DG), and combinations thereof. In some embodiments, oligomycin is added at 10 mM. In some embodiments, FCCP is added at 10 mM. In some embodiments, rotenone is added at 2.5 mM. In some embodiments, antimycin A is added at 2.5 mM. In some embodiments, 2-deoxyglucose (2-DG) is added at 500 mM. In some embodiments, glycolytic capacity, glycolytic reserve, and / or non-glycolytic acidification are measured. Typically, TILs have a glycolytic reserve of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 80% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 90% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 95% to about 99% of the basal respiration rate of freshly harvested TILs.

[0248]

[0260] In some embodiments, the metabolic assay is basal glycolysis. In some embodiments, the second expanded TILs or additional second expanded TILs, e.g., those at step D (e.g., including TILs that have undergone additional second expansion, designated reREP), have an increase in basal glycolysis of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. In some embodiments, the second expanded TILs or additional second expanded TILs, e.g., those at step D (including TILs designated reREP TILs), have an increase in basal glycolysis of about 2-fold to about 10-fold. In some embodiments, the second expanded TILs or additional second expanded TILs, e.g., those at step D (including TILs designated reREP TILs), have an increase in basal glycolysis of about 2-fold to about 10-fold. In some embodiments, the second expansion culture of TILs or additional second expansion cultures, such as those in step D (reREP), exhibits an increase in basal glycolysis of about 2-fold to about 8-fold. In some embodiments, the second expanded TILs or additional second expanded cultures, e.g., those at Step D (including TILs referred to as reREP TILs), increase basal glycolysis by about 3-fold to about 7-fold. In some embodiments, the second expanded TILs or additional second expanded cultures, e.g., those at Step D (including TILs referred to as reREP TILs), increase basal glycolysis by about 2-fold to about 4-fold. In some embodiments, the second expanded TILs or additional second expanded cultures, e.g., those at Step D (including TILs referred to as reREP TILs), increase basal glycolysis by about 2-fold to about 3-fold.

[0249]

[0261] Generally, a second expansion of TIL or an additional second expansion, e.g., step Group D TILs (e.g., including TILs that have undergone an additional second expansion, designated reREP) have a glycolytic reserve of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 50% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 60% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 70% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 80% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 90% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the glycolytic reserve is about 95% to about 99% of the basal respiration rate of freshly harvested TILs. In some embodiments, the second expanded TILs or second additional expanded TILs (e.g., including TILs referred to as reREP TILs, such as those described in step D of FIG. 11 ) have a spare respiratory capacity that is not statistically significantly different from the basal respiration rate of freshly harvested TILs.

[0250]

[0262] Granzyme B production: Granzyme B enhances the ability of TILs to kill target cells. Granzyme B levels were also measured in culture supernatants restimulated as described above with antibodies against CD3, CD28, and CD137 / 4-1BB using the Human Granzyme B DuoSet ELISA Kit (R & D Systems, Minneapolis, MN). The TILs were used and evaluated according to the manufacturer's instructions. In some embodiments, the second expanded TILs or second additional expanded TILs (e.g., including TILs designated reREP TILs, such as those described in step D of FIG. 11 ) have increased granzyme B production. In some embodiments, the second expanded TILs or second additional expanded TILs (e.g., including TILs designated reREP TILs, such as those described in step D of FIG. 11 ) have increased cytotoxic activity.

[0251]

[0263] In some embodiments, the method comprises: , and assays for assessing TIL viability. In some embodiments, TILs are expanded as discussed above, including, for example, as provided in FIG. 11 . In some embodiments, TILs are cryopreserved prior to assessment of viability. In some embodiments, viability assessment includes thawing the TILs prior to performing the first expansion, the second expansion, and the additional second expansion. In some embodiments, the method provides assays for assessing cell proliferation, cytotoxicity, cell death, and / or other viability-related features of a TIL population. Viability can be measured by any of the TIL metabolic assays described above as well as any method known in the art for assessing cell viability. In some embodiments, the method provides assays for assessing cell proliferation, cytotoxicity, cell death, and / or other viability-related features of TILs expanded using the methods described herein, including as illustrated in FIG. 11 . do.

[0252]

[0264] The present invention also provides an assay method for determining TIL viability. The disclosure provides a method for assaying tumor infiltrating lymphocyte (TIL) viability by expanding TIL into a larger TIL population, the method comprising: (i) Obtaining a first population of pre-expanded TILs; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold or 100-fold more numerous than the second TIL population, and wherein the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population, and wherein the third population is further assayed for viability. Includes.

[0253]

[0265] In some embodiments, the method comprises: (iv) further comprising performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger TIL population than that obtained in step (iii), wherein the larger TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population, and wherein the third population is further assayed for viability.

[0254]

[0266] In some embodiments, prior to step (i), the cells are cryopreserved.

[0255]

[0267] In some embodiments, the cells are thawed prior to performing step (i).

[0256]

[0268] In some embodiments, to obtain enough TILs for analysis, steps ( iv) is repeated 1 to 4 times.

[0257]

[0269] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 It will be carried out within a period of 0 to approximately 50 days.

[0258]

[0270] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 It will be carried out within a period of 2 to approximately 48 days.

[0259]

[0271] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 It will be carried out within a period of 2 to approximately 45 days.

[0260]

[0272] In some embodiments, steps (i)-(iii) or (iv) are performed for about 4 This will be carried out within four days.

[0261]

[0273] In some embodiments, the cells from step (iii) or (iv) They express CD4, CD8, and TCRαβ to the same extent as freshly harvested cells.

[0262]

[0274] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). .

[0263]

[0275] In some embodiments, the PBMCs are 9 to 17 It is added to the cell culture on either day 1 or day 2.

[0264]

[0276] In some embodiments, the enzymes in the larger TIL population of step (iv) The effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0265]

[0277] In some embodiments, effector T cells and / or central memory -T cells exhibit increased CD57 expression and decreased CD56 expression.

[0266]

[0278] In some embodiments, the APC is an artificial APC (aAPC).

[0267]

[0279] In some embodiments, the method comprises administering to a subject a nucleic acid encoding a high affinity T cell receptor. The method further comprises transducing the first TIL population with an expression vector comprising the acid.

[0268]

[0280] In some embodiments, the transduction step occurs before step (i). do.

[0269]

[0281] In some embodiments, the method comprises administering to a subject a subject a T cell signaling molecule comprising at least one of: The method further comprises transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to one endodomain.

[0270]

[0282] In some embodiments, the transduction step occurs before step (i). do.

[0271]

[0283] In some embodiments, the TILs are assayed for viability.

[0272]

[0284] In some embodiments, the TILs are assayed for viability after cryopreservation. can be.

[0273]

[0285] In some embodiments, the TILs are cryopreserved and after step (iv). Assayed for viability.

[0274]

[0286] According to the present disclosure, methods for assaying the viability of TILs and / or administering them to a subject In some embodiments, the method for assaying tumor infiltrating lymphocytes (TILs) comprises: (i) Obtaining the first TIL population; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population; (iv) harvesting, washing, and cryopreserving the third TIL population; (v) cryopreserved TILs are stored at cryogenic temperatures; (vi) thawing the third TIL population to provide a thawed third TIL population; and (vii) performing an additional second expansion of a portion of the thawed third TIL population for a reREP period of at least 3 days by adding IL-2, OKT-3, and APC to the cell culture medium of the third population, wherein the third expansion is performed to obtain a fourth TIL population, and comparing the number of TILs in the fourth TIL population with the number of TILs in the third TIL population to determine a ratio; (viii) determining whether the thawed TIL population is suitable for administration to a patient based on the ratio of step (vii); (ix) administering a therapeutically effective dose of the thawed third TIL population to the patient when the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is determined to be greater than 5:1 in step (viii). Includes.

[0275]

[0287] In some embodiments, the reREP period is the number of TILs in the fourth TIL population. The procedure is continued until the ratio of the number of TILs in the first TIL group to the number of TILs in the third TIL group is greater than 50:1.

[0276]

[0288] In some embodiments, a number of TILs sufficient to provide a therapeutically effective dosage. is approximately 2.3 x 10 10 ~Approx. 13.7×10 10 There are individuals.

[0277]

[0289] In some embodiments, steps (i)-(vii) are carried out over a period of about 40 days to about 50 days. In some embodiments, steps (i)-(vii) are performed within a period of about 42 days to about 48 days. In some embodiments, steps (i)-(vii) are performed within a period of about 42 days to about 45 days. In some embodiments, steps (i)-(vii) are performed within about 44 days.

[0278]

[0290] In some embodiments, the cells from step (iii) or (vii) , CD4, CD8, and TCRαβ to the same extent as freshly harvested cells. In some embodiments, the cells are TILs.

[0279]

[0291] In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture in step (iii) on any day between 9 and 17.

[0280]

[0292] In some embodiments, the greater of step (iii) or (vii) The effector T cells and / or central memory T cells in the TIL population exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

[0281]

[0293] In some embodiments, effector T cells and / or central memory -T cells exhibit increased CD57 expression and decreased CD56 expression.

[0282]

[0294] In some embodiments, the APC is an artificial APC (aAPC).

[0283]

[0295] In some embodiments, the gene encoding the high affinity T cell receptor is Transducing a first population of TILs with the present vector.

[0284]

[0296] In some embodiments, the transduction step occurs before step (i). do.

[0285]

[0297] In some embodiments, at least one end of a T cell signaling molecule Transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to a CAR domain.

[0286]

[0298] In some embodiments, the transduction step occurs before step (i). do.

[0287]

[0299] In some embodiments, the TILs are evaluated for viability after step (vii). Assayed.

[0288]

[0300] The present disclosure also provides additional methods for assaying TILs. The present disclosure provides a method for assaying TILs, the method comprising: (i) Obtaining a portion of the first cryopreserved TIL population; (ii) thawing a portion of the first cryopreserved TIL population; (iii) performing a first expansion culture by culturing a portion of the first TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for a reREP period of at least 3 days to generate a second TIL population, wherein the portion from the first TIL population is compared with the second TIL population to determine a ratio of the number of TILs, and the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to a patient based on the ratio of step (iii); (v) determining that the first TIL population is suitable for use in therapeutic administration when the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1 in step (iv). Includes.

[0289]

[0301] In some embodiments, the number of TILs in the second TIL population is compared to the number of TILs in the first TIL population. The ratio of TIL numbers in some groups is greater than 50:1.

[0290]

[0302] In some embodiments, the method comprises any of the embodiments provided herein. and performing expansion of the entire first cryopreserved TIL population from step (i) by the method as described in 2.

[0291]

[0303] In some embodiments, the method further comprises the step of: It further includes administering the entire TIL population to the patient.

[0292]

[0304] In some embodiments, the cryopreserved TILs are thawed and a second expansion culture is performed. This determines whether the cells are sufficiently expanded. If the cells are expanded to a ratio of at least 5:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 10:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 15:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 20:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 25:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 30:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 35:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 40:1, the TILs are sufficiently viable for administration to a patient. If the cells are expanded to a ratio of at least 45:1, the TILs are sufficiently viable for administration to a patient. When the cells are expanded to a ratio of at least 5:1, the TILs are fully viable for administration to patients.

[0293]

[0305] The present disclosure also provides additional methods for assaying TILs. The present disclosure provides a method for assaying TILs, the method comprising: (i) Obtaining a portion of the first cryopreserved TIL population; (ii) thawing a portion of the first cryopreserved TIL population; (iii) performing a first expansion culture by culturing a portion of the first TIL population in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for a reREP period of at least 3 days to generate a second TIL population, wherein the portion from the first TIL population is compared with the second TIL population to determine a ratio of the number of TILs, and the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to the patient based on the ratio of step (iii); and (v) when the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1 in step (iv), therapeutically administering the remainder of the first TIL population to the patient. Includes.

[0294]

[0306] In some embodiments, the number of TILs in the second TIL population is compared to the number of TILs in the first TIL population. The ratio of TIL numbers in some groups is greater than 50:1.

[0295]

[0307] In some embodiments, the method comprises the steps of any of the methods of the preceding claims. and performing an expansion culture of the entire first cryopreserved TIL population from step (i).

[0296]

[0308] In some embodiments, the method further comprises the step of: It further includes administering the entire TIL population to the patient.

[0297]

[0309] In some embodiments, the method comprises assessing the metabolic health of the second TIL population. The method further includes the step of:

[0298]

[0310] In some embodiments, the method comprises assessing the phenotype of the second TIL population. The method further includes the steps of:

[0299]

[0311] In some embodiments, the antigen-presenting cells are allogeneic peripheral blood mononuclear cells.

[0300] L. Patient Treatment Methods

[0312] The treatment method begins with the initial collection and culture of TILs. Both of these methods have been described in the art, for example, by Jin et al. (J. Immunotherapy, 2012, 35(3):283-292) (incorporated herein by reference in its entirety). and throughout the Examples section below.

[0301]

[0313] The present invention provides a novel method for generating TILs that has not been described before, e.g., The present invention provides TILs produced by steps A-F. Expanded TILs as produced by steps A-F above or as otherwise described herein find particular use in treating cancer patients. For general methods of using TILs to treat cancer, see Goff, et al., J. Clinical Oncology, 2016, 34(20):2389-239, as well as the supplemental content; the entire (2003, 26:332-342; incorporated herein by reference in its entirety). Similarly, TILs produced according to the present invention can also be used to treat cancer. In some embodiments, TILs are grown from deposited resections of metastatic melanoma as previously described (see Dudley, et al., J. Immunother., 2003, 26:332-342; incorporated herein by reference in its entirety). Fresh tumors can be excised under sterile conditions. Representative samples can be collected for formal pathological analysis. mm 3 ~3mm 3 In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained per patient. In some embodiments, 20, 25, or 30 samples are obtained per patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained per patient. In some embodiments, 24 samples are obtained per patient. Samples may be placed in individual wells of a 24-well plate, maintained in growth medium containing high-dose IL-2 (6,000 IU / mL), and monitored for tumor destruction and / or TIL proliferation. Any tumors with viable cells remaining after treatment may be enzymatically digested into a single cell suspension and cryopreserved as described herein.

[0302]

[0314] In some embodiments, phenotypic analysis (CD3, CD4, CD8, and CD Expanded TILs can be sampled for immunohistochemistry (56) and tested against autologous tumors, if available. TILs can be considered responsive if overnight coculture results in interferon gamma (IFN-γ) levels >200 pg / mL and twice background (Goff, et al., J Immunother., 2010, 33:840-847; see references herein for the entirety). (The text is hereby incorporated by reference in its entirety.) In some embodiments, cultures with evidence of auto-responsiveness or sufficient growth patterns may be selected for a second expansion culture (e.g., a second expansion culture as provided in step D of FIG. 11 ), including a second expansion culture, sometimes referred to as a rapid expansion culture (REP). In some embodiments, expanded TILs with high auto-responsiveness (e.g., high proliferation during the second expansion culture) are selected for an additional second expansion culture. In some embodiments, TILs with high auto-responsiveness (e.g., high proliferation during the second expansion culture as provided in step D of FIG. 11 ) are selected for an additional second expansion culture according to step D of FIG. 11 .

[0303]

[0315] In some embodiments, the patient is not directly transferred to ACT (adoptive cell transfer), For example, in some embodiments, after tumor harvest and / or the first expansion, the cells are not utilized immediately. In such embodiments, the TILs can be cryopreserved and thawed two days before the second expansion step (e.g., in some embodiments, two days before the step referred to as the REP step). In such embodiments, the TILs can be cryopreserved and thawed two days before the second expansion step (e.g., in some embodiments, two days before step D as provided in FIG. 11). As described in various embodiments throughout this application, the second expansion (including the process referred to as REP) involves the use of OKT3 (anti-CD3) antibody (Miltenyi Biotech, San Diego, CA) and IL-2 (3,000 IU) in the presence of irradiated feeder cells (autologous, if possible) at a 100:1 ratio. / mL; Prometheus, San Diego, CA) was used (see Dudley, et al., J Immunother., 2003, 26:332-342; incorporated herein by reference in its entirety). In some embodiments, the TILs can be cryopreserved and thawed 5 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 4 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 3 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 2 days before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed 1 day before the second expansion step. In some embodiments, the TILs can be cryopreserved and thawed immediately before the second expansion step.

[0304]

[0316] The cell phenotype of the cryopreserved TIL samples from the transfusion bag was determined by the surface markers CD3, C Serum cytokines were measured by flow cytometry (FlowJo) for CD4, CD8, CCR7, and CD45RA (BD BioSciences) and by any of the methods described herein. Serum cytokines were measured using standard enzyme-linked immunosorbent assays. Elevated serum IFN-γ was defined as >100 pg / mL and greater than 4 cm baseline levels.

[0305] 1. Optional lymphodepletion preconditioning of patients

[0317] Experimental findings suggest that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes may be beneficial for the treatment of It has been noted that removal of nodal T cells and competing elements of the immune system ("cytokine sinks") plays an important role in enhancing therapeutic efficacy. Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") in patients prior to introducing the second expanded TILs of the present invention or a second additional expanded TILs (e.g., such as those described in step D of FIG. 11, including TILs referred to as reREP TILs).

[0306]

[0318] Generally, lymphodepletion is performed with fludarabine and / or cyclophosphamide (the The active form is called mafosfamide) and combinations thereof. Such methods are described in Gassner et al. (Cancer Immunol Immunother. 2011, 60(1):75-85 , Muranski, et al., Nat Clin Pract Oncol., 2006 3(12):668-681, Dudley, et al., J Clin Oncol 2008, 26:5233-5239, and Dudley, et al., J Clin Oncol. 2005, 23(10):2346-2357, all of which are incorporated by reference herein in their entireties.

[0307]

[0319] In some embodiments, fludarabine is 0.5 μg / ml to 10 μg / ml In some embodiments, the fludarabine is at a concentration of 1 μg / ml fludarabine (Sigma-Aldrich, MO, USA). In some embodiments, the fludarabine treatment is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, fludarabine is administered at a dosage of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day. In some embodiments, the fludarabine treatment is at 35 mg / kg / day for 2-7 days. In some embodiments, the fludarabine treatment is at 35 mg / kg / day for 4-5 days. In some embodiments, the fludarabine treatment is at 25 mg / kg / day for 4-5 days.

[0308]

[0320] In some embodiments, mafosfamide, an active form of cyclophosphamide, is used. In some embodiments, the active form of cyclophosphamide, mafosfamide, is at a concentration of 1 μg / ml. In some embodiments, cyclophosphamide treatment is for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, cyclophosphamide is at a concentration of 100 mg / ml. 2 / day, 150mg / m 2 / day, 175mg / m 2 / day, 200mg / m 2 / day, 225mg / m 2 / day, 250mg / m 2 / day, 275mg / m 2 / day, or 300 mg / m 2 In some embodiments, cyclophosphamide is administered at a dosage of 35 mg / kg / day for 2-7 days. In some embodiments, cyclophosphamide is administered at a dosage of 250 mg / kg / day. In some embodiments, cyclophosphamide is administered intravenously (i.e., iv). In some embodiments, cyclophosphamide treatment is at 35 mg / kg / day for 2-7 days. In some embodiments, cyclophosphamide treatment is at 250 mg / kg / day. 2 / day iv for 4-5 days. In some embodiments, cyclophosphamide treatment is 250 mg / m 2 / day iv for 4 days.

[0309]

[0321] In some embodiments, fludarabine and cyclophosphamide are administered to the patient simultaneously. In some embodiments, fludarabine is administered at 25 mg / m for 4 days. 2 / day, given intravenously, and cyclophosphamide at 250 mg / m 2 / day, given intravenously.

[0310]

[0322] This protocol involves administering fludarabine (25 mg / m 2 / day, iv) and Clofosfamide (250 mg / m 2 / day, iv) for 4 days.

[0311] 2. Exemplary Treatment Embodiments

[0323] In some embodiments, the present disclosure provides a method for the treatment of cancer by a tumor-infiltrating lymphocyte (TIL) population. (b) obtaining a second TIL population by performing an initial expansion culture of the first TIL population in a first cell culture medium, wherein the second TIL population is at least 5-fold more numerous than the first TIL population, and wherein the first cell culture medium comprises IL-2; (c) obtaining a third TIL population by performing a rapid expansion culture of the second TIL population using a myeloid artificial antigen presenting cell (myeloid aAPC) population in a second cell culture medium, wherein 7 days after the initiation of the rapid expansion culture, the third TIL population is at least 50-fold more numerous than the second TIL population; and wherein the second cell culture medium comprises IL-2 and OKT-3; and (d) administering a therapeutically effective amount of the third TIL population to a patient with cancer. In some embodiments, IL-2 is present in the second cell culture medium at an initial concentration of about 3000 IU / mL, and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL. In some embodiments, the first expansion culture is performed for a period of 14 days or less. In some embodiments, the first expansion culture is performed using a gas-permeable container. In some embodiments, the second expansion culture is performed using a gas-permeable container. In some embodiments, the ratio of the second TIL population to the aAPC population in the rapid expansion culture is 1:80 to 1:400. In some embodiments, the ratio of the second TIL population to the aAPC population in the rapid expansion culture is about 1:300. In some embodiments, the cancer being treated 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, renal cancer, and renal cell carcinoma. In some embodiments, the cancer being treated is selected from the group consisting of melanoma, ovarian cancer, and cervical cancer. In some embodiments, the cancer being treated is melanoma. In some embodiments, the cancer being treated is ovarian cancer. In some embodiments, the cancer being treated is cervical cancer. In some embodiments, the method of treating cancer further comprises treating the patient with a non-myeloablative lymphodepletion regimen before administering the third population of TILs to the patient.In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by administering fludarabine at a dose of 25 mg / m2 / day for five days. In some embodiments, the high-dose IL-2 regimen comprises 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours to a tolerated dose.

[0312] 3. Co-administration Method

[0324] In some embodiments, the method is performed as described in steps A through F herein. The TILs produced can be administered in combination with one or more immune checkpoint modulators, such as the antibodies described below. For example, antibodies that target PD-1 and can be co-administered with the TILs of the present invention include, but are not limited to, nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), humanized Anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR -042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-P D-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is from clone: ​​RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Other suitable antibodies suitable for use in the co-administration method with TILs generated by steps A-F as described herein is an anti-PD-1 antibody disclosed in U.S. Patent No. 8,008,449 (incorporated herein by reference). In some embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibody known in the art that binds to PD-L1 to disrupt the interaction between PD-1 and PD-L1 and stimulates an anti-tumor immune response is suitable for use in the co-administration method with TILs generated by steps A-F as described herein. For example, antibodies targeting PD-L1 in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies targeting PD-L1 are disclosed in U.S. Patent No. 7,943,743, incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the co-administration method with TILs produced by steps A-F as described herein. In some embodiments, subjects receiving the combination of TILs produced by steps A-F are co-administered with an anti-PD-1 antibody when the patient has a cancer type that is refractory to administration of an anti-PD-1 antibody alone. In some embodiments, when the patient has refactory melanoma, the patient receives TILs in combination with an anti-PD-1 antibody. In some embodiments, when the patient has non-small cell lung cancer (NSCLC), the patient is administered TILs in combination with anti-PD-1.

[0313] 4. Adoptive cell transfer Adoptive cell transfer (ACT) is a highly effective form of immunotherapy that involves the transfer of immune cells with anti-tumor activity into cancer patients. ACT is a therapeutic approach that involves the in vitro identification of lymphocytes with anti-tumor activity, their in vitro expansion to increase their numbers, and their infusion into cancer-bearing hosts. Lymphocytes used for adoptive transfer may be derived from the stroma of resected tumors (tumor-infiltrating lymphocytes or TILs). TILs for ACT can be prepared as described herein. In some embodiments, TILs are prepared, for example, by a method as described in FIG. 11. TILs can also be derived from blood if they are genetically modified to express anti-tumor T cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched in mixed lymphocyte tumor cell cultures (MLTCs), or cloned using autologous antigen-presenting cells and tumor-derived peptides. ACT in which lymphocytes are derived from the cancer-bearing host to be infused is referred to as autologous ACT. U.S. Patent Application Publication No. 2011 / 0052530 relates to methods of implementing adoptive cell therapy to promote cancer regression, primarily for the treatment of patients with metastatic melanoma (incorporated by reference in its entirety with respect to these methods).

[0314]

[0002] In some embodiments, TILs can be administered as described herein. In some embodiments, TILs can be administered in a single dose. Such administration may be by injection, for example, intravenous injection. In some embodiments, TILs and / or cytotoxic lymphocytes may be administered in multiple doses. Administration may be once, twice, three times, four times, five times, six times, or more than six times per year. Administration may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs and / or cytotoxic lymphocytes may be continued for as long as necessary.

[0315] I. Illustrative Embodiments

[0325] In one embodiment, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs). The present invention provides a method for performing a method for detecting a defect, the method comprising: (a) Obtaining a first population of TILs from a tumor resected from a patient; (b) obtaining a second TIL population by performing an initial expansion culture of the first TIL population in a first cell culture medium, wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second TIL population, wherein a third TIL population is at least 100-fold more numerous than the second TIL population; and the second cell culture medium comprises IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), and the rapid expansion culture is performed for at least 14 days; (d) removing the cells from the second cell culture medium and, optionally, cryopreserving the cells in a storage medium to obtain a third cell population; (e) optionally thawing the third cell population; and (f) performing a second rapid expansion of the third TIL population in a third cell culture medium, the third cell culture medium comprising IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), and the second rapid expansion is performed for at least 14 days, thereby obtaining a fourth TIL population, the fourth cell population exhibiting increased effector T cell and / or central memory T cell subpopulations relative to the second TIL population; and g) optionally repeating step f) one or more times. Includes.

[0316]

[0326] In one embodiment, the present invention relates to a method for treating re-stimulated cells comprising administering to a subject the re-stimulated cells a CD4, CD8 and express TCRαβ to the same extent as freshly harvested cells.

[0317]

[0327] In one embodiment, the present invention relates to a method for producing a reREP medium containing peripheral blood mononuclear cells (PBMCs). BMC) is provided.

[0318]

[0328] In one embodiment, the present invention relates to a method for detecting PBMCs on any one of days 9 to 17. In some embodiments, the invention provides that the PBMCs are added to the TILs on days 9, 10, 11, 12, 13, 14, 15, 16, and / or 17.

[0319]

[0329] In one embodiment, the present invention relates to a method for treating atopic dermatitis, wherein the reREP medium contains aAPC. and provide.

[0320]

[0330] In one embodiment, the present invention provides a method for detecting high-affinity T cell receptors in cryopreserved TILs. The vector is transfected with an expression vector containing a nucleic acid encoding the vector.

[0321]

[0331] In one embodiment, the present invention provides a method for detecting T cell signaling in cryopreserved TILs. The present invention provides a method for the treatment of cancer, comprising transfecting a subject with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an immunoglobulin light chain fused to an endodomain of the molecule.

[0322]

[0332] In one embodiment, the present invention provides that the restimulated TILs are infused into the patient. provide.

[0323]

[0333] In one embodiment, the present invention provides a method for preparing a cell culture medium comprising the steps of: The method further comprises removing the cells.

[0324]

[0334] In one embodiment, the present invention provides a method for producing a TIL sufficient for a therapeutic dosage of the TIL. provided that step f) is repeated a sufficient number of times to obtain

[0325]

[0335] In one embodiment, the present invention provides a method for the treatment of a cancer by the methods described above and herein. The present invention provides a restimulated TIL population generated by the method of the present invention.

[0326]

[0336] In one embodiment, the present invention provides a method for producing a composition comprising: A population of restimulated TILs is provided, wherein said restimulated TILs have at least a two-fold increase in basal glycolysis when compared to said thawed cryopreserved TILs.

[0327]

[0337] In one embodiment, the present invention provides a method for assessing the metabolic activity of a TIL cell population. The present invention provides a method for measuring basal glycolysis of the cells.

[0328]

[0338] In one embodiment, the present invention provides a method for assessing the metabolic activity of a TIL cell population. A method is provided which comprises measuring the basal respiration of said cells.

[0329]

[0339] In one embodiment, the present invention provides a method for assessing the metabolic activity of a TIL cell population. The present invention provides a method for measuring the spare respiratory capacity (SRC) of said cells.

[0330]

[0340] In one embodiment, the present invention provides a method for assessing the metabolic activity of a TIL cell population. The present invention provides a method for determining the glycolytic reserve of the cells.

[0331]

[0341] In one embodiment, the present invention provides a method for the treatment of tumors caused by a tumor-infiltrating lymphocyte (TIL) population. and a method for treating cancer in a patient, the method comprising: a) obtaining a primary TIL population from said patient; b) rapidly expanding the primary TIL population to form an expanded TIL population; c) cryopreserving the expanded population to form a cryopreserved TIL population; d) thawing the cryopreserved TIL population; e) culturing the cryopreserved TIL population in a medium containing IL-2 and an anti-CD3 antibody to form a reREP TIL population; and f) administering a therapeutically effective amount of reREP TIL cells to said patient. Includes.

[0332]

[0342] In one embodiment, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs). The present invention provides a method for performing a method for detecting a defect, the method comprising: (a) Obtaining a first population of TILs from a tumor resected from a patient. (b) obtaining a second TIL population by performing an initial expansion culture of the first TIL population in a first cell culture medium, wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second TIL population, wherein the third TIL population is at least 100-fold more numerous than the second TIL population; and the second cell culture medium comprises IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), and the rapid expansion is performed for at least 14 days; (d) removing the cells from the second cell culture medium and, optionally, cryopreserving the cells in a storage medium to obtain a third cell population; (e) optionally thawing the third cell population; (f) performing a second rapid expansion of the third TIL population in a third cell culture medium, the third cell culture medium comprising IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs), and the second rapid expansion is performed for at least 14 days, thereby obtaining a fourth TIL population, the fourth cell population exhibiting increased effector T cell and / or central memory T cell subpopulations relative to the second TIL population; and (g) administering a therapeutically effective amount of reREP TIL cells to said patient. Includes.

[0333]

[0343] In one embodiment, the present invention provides a method for preparing a cell culture medium comprising the steps of: The method further comprises removing the cells.

[0334]

[0344] In one embodiment, the present invention provides a method for producing a TIL sufficient for a therapeutic dosage of the TIL. provided that step f) is repeated a sufficient number of times to obtain [Example]

[0335] Example Example 1: Restimulation Protocol

[0345] As discussed herein, after harvesting or thawing TILs grown in REP, A restimulation protocol and assay was developed using fresh antigen restimulation.

[0336]

[0346] The purpose of this example is to demonstrate the ability of post-REP tumor-infiltrating lymphocytes in restimulation assays. The objective of this study was to test the proliferation / expansion of post-REP TILs (TILs after step D according to Figure 11) by allogeneic PBMC feeder cells, anti-CD3 (clone OKT3) antibody, and interleukin-2 (IL-2). Viable cells were counted and recorded on day 7.

[0337]

[0347] Pre-lymphocyte depletion to enhance TIL survival and expansion in vivo Post-REP TILs (TILs after step D according to Figure 11) were infused into patients who had received the treatment. When the TILs were reinfused into the patient, they encountered antigen, resulting in their activation, but the TILs were ultimately short-lived. In ACT, restimulation of TILs through antigen contact and exposure to IL-2 can result in TIL proliferation and tumor control, or, due to the lack of appropriate costimulation, can lead to apoptosis-mediated elimination (activation-induced cell death) or induction of a non-proliferative (anergic) state. Without being bound by theory, restimulation of post-REP TILs with allogeneic PBMC feeder cells (e.g., restimulation of TILs after step D according to Figure 11) can mimic this in vivo process by providing antigen stimulation and the necessary cytokines for TIL expansion. Post-REP TILs (TILs after step D according to Figure 11) were activated in the REP flask via membrane receptors on the feeder MNCs that bind to anti-CD3 (clone OKT3) antibodies and crosslink to TILs, stimulating them to expand.

[0338] Proliferation / expansion of post-REP tumor-infiltrating lymphocytes in restimulation assays

[0348] Post-REP (TILs after step D according to Figure 11) TILs were cultured in allogeneic PB The cells were restimulated with MC feeder cells, anti-CD3 (clone OKT3) antibody, and interleukin-2 (IL-2). On day 7, viable cells were counted and recorded.

[0339]

[0349] In some embodiments, this procedure also includes testing of the current REP protocol. It can also be applied to validation.

[0340] [Table 3]

[0341] [Table 4]

[0342] [Table 5]

[0343]

[0350] Prior lymphocyte depletion to promote TIL survival and expansion in vivo Post-REP (TILs after step D according to Figure 11) TILs were infused into patients who had previously undergone ACT. When the TILs were reinfused into the patient, they encountered antigen, resulting in TIL activation, but the TILs were ultimately short-lived. In ACT, restimulation of TILs through antigen contact and exposure to IL-2 can result in TIL proliferation and tumor control, or, due to the lack of appropriate costimulation, can lead to elimination via apoptosis (activation-induced cell death) or induction of a non-proliferative (anergic) state. Our hypothesis was that restimulation of post-REP TILs with allogeneic PBMC feeder cells would mimic this in vivo process by providing antigen stimulation and the necessary cytokines for TIL expansion. Post-REP TILs were grown in REP flasks on feeder cells that bound to anti-CD3 (clone OKT3) antibodies and crosslinked to TILs, stimulating them to expand. -activated via membrane receptors on MNCs.

[0344] procedure

[0351] Fresh post-REP (TILs after step D according to Figure 11) or thawed frozen Post-REP (TILs after step D according to Figure 11) TILs were washed once with CM1 medium. 2 x 10 TILs were plated in CM2 in a 24-well tissue culture plate. 6 MNC feeder cells, 30 ng / ml OKT3, 1 x 10 4 Re-REP (repeat of step D according to Figure 11) was set up with 100 post-REP TILs + 3,000 IU / ml rhIL-2. Cultures were incubated for 7 days in a humidified incubator at 37°C with 5% CO2, at which point viable cell recovery and viability were determined. Fold expansion of TILs was calculated based on viable cell counts. ReREP-Day 0 1. Preparing TILs TILs were obtained from fresh or frozen post-REP. TIL cultures were removed from the incubator and transferred to a BSC. 200 μl was then removed and cells were counted using a Cellometer K2. Counts were recorded.

[0345] Prepare feeder cells

[0352] This protocol requires a minimum of 20 × 10 6 feeder cells were required. Each 1 ml vial frozen by SDBB ​​contains 100 x 10 6 Assuming a 50% recovery rate upon thawing from LN2 storage, each REP contained an estimated 100 x 10 viable cells. 6 Given viable cells, it was recommended to thaw at least two vials of feeder cells per lot. Prior to thawing the feeder cells, approximately 50 ml of CM2 without rhIL-2 was pre-warmed for each feeder lot to be tested. The designated feeder lot vials were removed from LN2 storage and placed on ice. The vials were transferred to the tissue culture room. The vials were thawed in a 37°C water bath. The vials were transferred to the BSC and sprayed or wiped with 70% EtOH or IPA. Using a transfer pipette, the contents of the feeder vial were immediately transferred into 50 mL of warm CM2 in a 50 mL conical tube. 200 μl was removed and the cells were counted using a Cellometer K2. The count was The cells were centrifuged at 350 × g for 10 minutes and then incubated at 2 × 10 in warm CM2 + 3000 IU / ml rhIL-2. 6 Aspirate the supernatant and resuspend cells in the desired volume in cells / ml.

[0346] Prepare a CM2+ 3000 IU / ml working solution

[0353] Prepare sufficient CM2 for the required conditions. Each well contained 6 × 10 CM2 containing 3000 IU / mL rhIL-2. 6From the IU / mL stock, 50 μl was required for each 100 ml of CM2.

[0347] Prepare MACS® GMP CD3 pure (OKT3) working solution

[0354] A stock solution of OKT3 (1 mg / ml) was removed from a 4°C refrigerator. A final concentration of 30 ng / ml OKT3 was used in the REP. 60 ng of OKT3 was required per 2 ml of CM2 medium in each 24-well plate. TIL + feeder, TIL alone, and feeder alone conditions were cultured in triplicate. For each feeder lot tested, 1000 μl of a 1:1000 dilution of 1 mg / ml OKT3 was made for a working concentration of 1 μg / ml (1,000 ng / ml). For 9 wells, 1000 μl of a 1:1000 dilution of 1 mg / ml OKT3 was made. 1 μl of 1 mg / ml OKT3 + 999 μl of CM2 containing 3,000 IU / ml IL-2 was made.

[0348] 1. Preparing 24-well Plates and Co-culturing

[0355] For each ReREP to be tested, 9 wells of a 24-well plate are required It was.

[0349]

[0356] Label each plate with the experiment name, feeder lot number, post-rep TIL name, Each plate was labeled with the date and operator's initials. Each plate was filled with the components listed in Table 8. Each component was added to fill each well with a total of 2 ml, and the plate was placed in a 37°C incubator. The plate was carefully mixed three times using a 1 ml pipette.

[0350] [Table 6]

[0351] Medium change - Day 5

[0357] CM2 was prepared with 3000 IU / ml rhIL-2. 10 ml was required. One ml of medium was removed from each well and discarded. Using a 1 ml pipette, 1 ml of warm CM2 containing 3000 IU / mL rhIL-2 was transferred to each well. The plate was returned to the incubator.

[0352] Recovery - Day 7

[0358] Remove cell clumps by mixing each well using a 1 ml serological pipette. After thoroughly mixing the cell suspension by pipetting, 200 μl was removed and the cells were counted using a Cellometer K2. All conditions were counted and recorded for TIL+OKT3 and feeder+OKT3.

[0353]

[0359] In addition to the 24-well ReREP, four upright T25 tissue culture flasks were M2 medium 1.3×10 7 MNC feeder cells, 30ng / ml OKT3, 0.65 x 10 5 Another reREP was set up with pre-REP TILs + 3,000 IU / ml rhIL-2. Note: See "Determination of Irradiated Allogeneic Feeder Cells for the LN-144 Rapid Expansion Protocol" (Example 6).

[0354]

[0360] Allocation of cells to functional assays:

[0355] [Table 7]

[0356] Judgment / Compliance Criteria

[0357] [Table 8]

[0358] Reference Procedures - Included in the Examples Below

[0359] [Table 9]

[0360] Example 2: Cell count and viability of TIL cultures using the Cellometer K2 cell counter decision

[0361] This example provides an illustrative description of how the operation of the Cellometer K2 Image Cytometer automated cell counter was performed.

[0361]

[0362] Scope of application: Determination of total cell number and viability in cell cultures.

[0362] [Table 10]

[0363] procedure Cell suspension preparation Trypan Blue Preparation The final trypan blue concentration was 0.1%. The manufacturer stocks 0.2%. When using trypan blue with the Cellometer K2, it is recommended to prepare a solution. Trypan blue (0.4%) was diluted to 0.2% in PBS. Trypan blue was filtered through a 0.2-0.4 micron filter and aliquoted into labeled tubes with caps. The cell suspension was mixed 1:1 with 0.2% trypan blue.

[0364] AOPI preparation AOPI solution was obtained when using AOPI on the Cellometer K2. Cell samples were stained 1:1 with AOPI solution. Note: When counting highly concentrated cultures, cell samples were diluted with cell culture medium before the final 1:1 dilution with trypan blue or AOPI. The manufacturer's recommended counting range was used to determine the best dilution to use.

[0365] Cellometer K2 Setup

[0365] The Cellometer K2 instrument was powered on. The Cellometer Image Cytometer icon was selected on the associated computer monitor. On the main software screen, one of the assays listed in the drop-down box was selected. When I selected Assay, the cell type and image mode were automatically imported. In the "Sample" section, I clicked Set User / Sample ID, which opened a separate screen to enter the operator's information for the specimen. I entered a "User ID," which consisted of the user's three initials. A "Sample ID" was entered. The sample ID was based on the specimen information received.

[0366] Set up the dilution parameters If no other dilutions were made except for the 1:1 mix, the dilution factor was 2. If a dilution was made before the final 1:1 mix, the dilution factor was 2x the previous dilution. The dilution factor was updated depending on the mix used.

[0367] Cell counting The plastic backing was removed from both sides of the Cellometer counting chamber slide (SD100) and placed on a clean, lint-free wipe. After preparing the cell suspension, a small aliquot of sample was removed and transferred to a well or tube in a multi-well cell culture plate. When diluting samples, dilutions were performed using cell culture medium. 20 μl of cell suspension was added to a well or tube in a multi-well cell culture plate. 20 μl of 0.2% trypan blue or AOPI solution was added to the 20 μl cell suspension, and the sample was mixed thoroughly. 20 μl of the 1:1 solution was measured and transferred to one side of the counting chamber. Note: The transparent area of ​​the slide was not touched. If necessary, the sample was repeated on the other side of the slide. The chamber was inserted into the slot on the front of the Cellometer. For AOPI cell counts, select "Preview Fl" on the main screen to preview the green fluorescent (live cell) image. For trypan blue counts, select "Preview Brightfield." Use the focusing wheel to bring the image into focus. The cells had a bright center and a clearly defined outline. Select "Count" to start the counting process. The counting results on the computer screen The results were displayed in a pop-up box, which showed the outcome of the counting process.

[0368] Example 3: Cellometer IC2 Image Cytometer Automated Cell Counter

[0368] This example describes the operating procedure for the Cellometer K2 Image Cytometer automated cell counter. 1.Definition μl microliter AOPI Acridine Orange Propidium Iodine BSC Biological Safety Cabinet DPBS Dulbecco's Phosphate Buffered Saline ml milliliter MNC mononuclear blood cell NA Not Applicable PBMC peripheral blood mononuclear cells PPE Personal Protective Equipment Initial TIL culture before rapid expansion protocol of pre-REP cultures REP Rapid Expansion Culture Protocol TIL tumor-infiltrating lymphocytes 7. Procedure 7.1 Cell suspension preparation 7.1.1 Trypan Blue Preparation The final trypan blue concentration was 0.1%. The manufacturer recommends preparing a 0.2% stock solution. 7.1.1.1 When using trypan blue in the Cellometer K2, use stock ( 0.4%) was diluted to 0.2% with PBS. 7.1.1.2 Trypan blue was filtered through a 0.2-0.4 micron filter and divided into small aliquots into labeled, capped tubes. 7.1.1.3 The cell suspension was mixed 1:1 with 0.2% trypan blue. 7.1.2 AOPI preparation 7.1.2.1 When using AOPI with the Cellometer K2, add AOPI solution. I did it. 7.1.2.2 Cell samples were stained 1:1 with AOPI solution. NOTE: When counting high concentration cultures, cell samples were diluted in cell culture medium before the final 1:1 dilution with trypan blue or AOPI. The manufacturer's recommended count range was used to determine the best dilution to use. 7.2 Cellometer K2 Setup 7.2.1 The Cellometer K2 instrument was turned on. 7.2.2 The Cellometer Image Cytometer icon was selected on the associated computer monitor. 7.2.3 On the main screen of the software, select one of the assays listed in the drop-down box. 7.2.3.1 Once the appropriate assay was selected, the cell type and image mode were automatically captured. 7.2.3.2 In the "Sample" section, select Set User / Sample ID to open a separate screen to enter operator information for the specimen. 7.2.3.2.1 "User ID" has been entered. 7.2.3.2.2 Entered the "Sample ID". The sample ID is Based on collected specimen information. 7.2.3.3 Dilution parameters were set up. 7.2.3.3.1 If no other dilutions were made except for a 1:1 mix, the dilution factor was 2. 7.2.3.3.2 If a dilution was made before the final 1:1 mix, the dilution factor was twice the previous dilution. 7.2.3.3.3 The dilution factor was updated according to the mixture used in the dilution section of the screen. Select the pencil icon to bring up the dialogue screen. 7.2.3.3.4 Confirmed that the Fl Image and F2 Image sections were identical to each other. 7.2.3.3.5 Selected the "Save" button after completing the setup. 7.3 Cell counting 7.3.1 Remove the plastic strips from both sides of the Cellometer Counting Chamber Slide (SD100). The backing of the mask was removed and placed on a clean lint-free wipe. 7.3.2 After preparation of the cell suspension, a small aliquot of the sample was removed and transferred to a well or tube of a multi-well cell culture plate. 7.3.3 When diluting samples, dilutions were performed using cell culture medium. 7.3.4 20 μl of cell suspension was added to the wells or tubes of a multi-well cell culture plate. 7.3.5 To 20 μl of cell suspension, 20 μl of 0.2% trypan blue or AOPI solution was added and the sample was mixed thoroughly. 7.3.6 Measure 20 μl of the 1:1 solution and transfer it to one side of the counting chamber. Note: The clear area of ​​the slide was not touched. 7.3.7 If necessary, the sample was repeated on the other side of the slide. 7.3.8. The chamber was inserted into the slot on the front of the Cellometer. 7.3.8 For AOPI cell counting, select "Preview Fl" on the main screen to preview the green fluorescent (live cell) image. For trypan blue counting, select "Preview Brightfield." 7.3.9 The image was brought into optimum focus using the focusing wheel. Cells had bright centers and clearly defined edges. 7.3.10 Select "Count" to start the counting process. 7.3.11 The results were displayed in a counting results pop-up box on the computer screen, which showed the outcome of the counting process.

[0369] Example 4: Preparation of IL-2 stock solution (CellGenix)

[0369] This example describes an exemplary procedure for preparing an IL-2 stock solution.

[0370]

[0370] Definition / Abbreviation μL: microliter or μl BSC: Biological Safety Cabinet BSL2: Biosafety Level 2 D-PBS: Dulbecco's phosphate buffered saline G: Gauge GMP: Good Manufacturing Processing (GMP) is a standard for manufacturing and quality control of pharmaceuticals and quasi-drugs. HAc: acetic acid HSA: human serum albumin mL: milliliter NA: Not applicable PPE: Personal Protective Equipment rhIL-2; IL-2: recombinant human interleukin-2 COA: Certificate of Analysis 6. Procedure 6.1 A 0.2% acetic acid solution (HAc) was prepared. 6.1.1 29 mL of sterile water was transferred to a 50 mL conical tube. 6.1.2 1 mL of 1N acetic acid was added to a 50 mL conical tube. 6.1.3 Mix thoroughly by inverting the tube 2-3 times. 6.1.4 Sterilize the HAc solution by filtration using a Steriflip filter. Ta. 6.1.5 The cap was replaced, the date was affixed, and the solution was labeled "Sterile 0.2% Acetic Acid Solution." 6.1.6 The solution expired after 2 months and was stored at room temperature. 6.2 1% HSA in PBS was prepared. 6.2.1 4 mL of 25% HSA stock solution was added to 96 mL PBS in a 150 mL sterile filter unit. 6.2.2 The solution was filtered. 6.2.3 The cap was closed, the date was added, and the solution was labeled "1% HSA in PBS." 6.2.4 The solution expired after 2 months and was stored at 4°C. 6.3 Documentation was prepared for each vial of rhIL-2 prepared. 6.4 rhIL-2 stock solution (6 × 10 6 IU / mL final concentration) was prepared. 6.4.1 rh1L-2 varied from lot to lot and required the following information to be included on the manufacturer's Certificate of Analysis (COA): 6.4.1.1 Mass of rhIL-2 per vial (mg) 6.4.1.2 rhIL-2 specific activity (IU / mg) 6.4.1.3 Recommended 0.2% HAc Reconstitution Volume (mL) 6.4.2 The volume of 1% HSA required for a rhIL-2 lot was calculated using the following formula:

number

number

[0371] Example 5: Media preparation for the pre-REP and REP processes

[0371] Examples include, but are not limited to, metastatic melanoma, head and neck squamous cell carcinoma, ovarian cancer, This paper describes the preparation of tissue culture medium used in protocols involving the culture of tumor-infiltrating lymphocytes (TILs) derived from various tumor types, including ripple-negative breast cancer and lung adenocarcinoma. In most cases, this medium was used for the preparation of any of the TILs described in this application and the Examples.

[0372]

[0372] Definition μg microgram μm micrometer μM micromolar AIM-V(R) Serum-free tissue culture medium (Thermo Fisher Scientific) BSC Biological Safety Cabinet CM1 Complete Medium #1 CM2 Complete Medium #2 CM3 complete medium #3 CM4 Complete Medium #4 IU or U International Unit ml milliliter mM millimolar concentration NA Not Applicable PPE Personal Protective Equipment Pre-REP Pre-Rapid Expansion Culture Process REP Rapid Expansion Process rhIL-2, IL-2 recombinant human interleukin-2 RPMI1640 Roswell Park Memorial Institute ) Culture medium, Formula 1640 SOP Standard Operating Procedure TIL tumor-infiltrating lymphocytes 7. Procedure 7.1 All procedures were performed using aseptic technique in a BSC (Class II, Type A2). 7.1.1 The surface of the hood was sprayed with 70% ethanol before use. 7.1.2 All materials and reagents were sprayed with 70% ethanol before being placed in the tissue culture hood. 7.2 Aliquoting 200mM L-glutamine 7.2.1 L-glutamine was supplied in a volume larger than required for serum preparation (e.g., 100 ml or 500 ml volumes). 7.2.2 A bottle of L-glutamine was thawed in a 37°C water bath. 7.2.3 L-glutamine precipitates after thawing, so mix the L-glutamine thoroughly after thawing, ensuring all precipitate goes back into solution before aliquoting. 7.2.4 Place a 5-10 ml aliquot of L-glutamine into a sterile 15 ml conical tube. 7.2.5 Label the tubes with the concentration, distributor, lot number, date of aliquoting, and expiration date. 7.2.6 Tubes were stored at -20°C and withdrawn for medium preparation as needed. 7.3 Preparation of CM1 7.3.1 The following reagents were removed from cold storage and warmed in a 37°C water bath: 7.3.1.1 RPMI1640 7.3.1.2 Human AB serum 7.3.1.3 200mM L-glutamine 7.3.2 The BME was removed from 4°C storage and placed in a tissue culture hood. 7.3.3 Gentamicin stock solution was placed in the tissue culture hood from room temperature storage. 7.3.4 CM1 medium was prepared by adding each of the components to the top of a 0.2 μm filter unit appropriate for the volume to be filtered according to Table 1 below.

[0373] [Table 11]

[0374] 7.3.5 CM1 medium bottles were labeled with the name, the preparer's initials, the date it was filtered / prepared, and a two-week expiration date, and stored at 4°C until needed for tissue culture. The medium was aliquoted into small volume bottles as needed. 7.3.6 Any remaining RPMI1640, human AB serum, or L-glutamine was stored at 4°C until the next medium preparation. 7.3.7 The BME stock bottles were returned to storage at 4°C. 7.3.8 The gentamicin stock bottle was returned to its appropriate room temperature storage location. 7.3.9 Due to the limited buffering capacity of the medium, CM1 was discarded within two weeks of preparation or upon phenol red pH indicator showing a significant shift in pH (bright red to pink color). 7.3.10 On the day of use, the required amount of CM1 was warmed in a 37°C water bath and 6000 IU / ml IL-2 was added. 7.3.11 Additional Additions - Optional 7.3.11.1 GlutaMAX® Supplemented to CM1 7.3.11.1.1 CM1 was prepared by substituting 2 mM GlutaMAX™ for 2 mM glutamine (final concentration, see Table 2). Once complete, the media bottle was supplemented and labeled "2 mM GlutaMAX" to prevent confusion with standard formulation CM1. 7.3.11.2 CM1 with additional antibiotic / antifungal 7.3.11.2.1 Some CM1 formulations required additional antibiotics or antifungals to prevent contamination of pre-REP TILs grown from certain tumor types. 7.3.11.2.2 Antibiotics / antimycotics were added to the final concentrations shown in Table 2 below. 7.3.11.2.3 Once completed, the media bottles were labeled by adding the name of the additional antibiotic / antimycotic to prevent confusion with the standard formulation CM1.

[0375] [Table 12]

[0376] 8.1 Preparation of CM2 8.1.1 Remove prepared CM1 from the refrigerator or prepare fresh CM1 according to the example above. 8.1.2 The AIM-V® was removed from the refrigerator. 8.1.3 Mix prepared CM1 with an equal volume of AIM-V® in a sterile media bottle. The required amount of CM2 was prepared by mixing the above ingredients. 8.1.4 On the day of use, 3000 IU / ml IL-2 was added to the CM2 medium. 8.1.5 On the day of use, a sufficient amount of CM2 containing 3000 IU / ml IL-2 was prepared. 8.1.6 CM2 medium bottles were labeled with the name, the preparer's initials, the date it was filtered / prepared, and a two-week expiration date and stored at 4°C until needed for tissue culture. The medium was aliquoted into small volume bottles as needed. 8.1.7 All CM2 without IL-2 was returned to the refrigerator where it was stored for up to two weeks or until the phenol red pH indicator showed a drastic shift in pH (bright red to pink color). 8.2 Preparation of CM3 8.2.1 CM3 was prepared on the day it was required for use. 8.2.2 CM3 is the same as AIM-V® medium and should be diluted to 3000 I on the day of use. U / ml IL-2 was added. 8.2.3 Adding IL-2 stock solution directly to the AIM-V bottle or bag A sufficient amount of CM3 was prepared for the experiment. It was mixed well by gently shaking. Immediately after adding it to the AIM-V, the bottle was labeled "3000 IU / ml IL-2." If there was excess CM3, it was stored at 4°C in bottles labeled with the medium name, the preparer's initials, the date the medium was prepared, and its expiration date (7 days after preparation). 8.2.4 Media supplemented with IL-2 was discarded after 7 days of storage at 4°C. 8.3 Preparation of CM4 8.3.1 CM4 was the same as CM3 plus 2 mM GlutaMAX™ (final concentration). 8.3.1.1 For every 1 L of CM3, 10 ml of 200 mM GlutaMAX™ was added. 8.3.2 Add IL-2 stock solution and GlutaMAX® (a brand name) to the AIM-V bottle or bag. A sufficient amount of CM4 for the experiment was prepared by directly adding the stock solution. Mix thoroughly by shaking gently. 8.3.3 Immediately after adding to the AIM-V, fill the bottle with "3000IL / nil IL-2 and GlutaMAX" label. 8.3.4 If there is excess CM4, it is stored at 4°C in bottles labeled with the medium name, "GlutaMAX", the preparer's initials, the date the medium was prepared, and its expiration date (7 days after preparation). 8.3.5 Media supplemented with IL-2 was discarded after 7 days of storage at 4°C.

[0377] Example 6: Evaluation of irradiated allogeneic feeder cells for the rapid expansion protocol of LN-144 This example demonstrates the isolation of individual loci from gamma-irradiated peripheral blood mononuclear cells (PBMCs, also known as MNCs). We describe a novel, simplified procedure for determining whether cells are eligible for use as allogeneic feeder cells in the exemplary methods described herein.

[0378] Each irradiated MNC feeder lot was prepared from an individual donor. Purified anti-CD3 Each lot or donor was individually screened for its ability to expand TILs in REP in the presence of (clone OKT3) antibody and interleukin-2 (IL-2). In addition, each lot of feeder cells was tested without the addition of TILs to ensure that the gamma radiation dose they received was sufficient to render them replication-incompetent. definition AOPI - Acridine Orange / Propidium Iodide ·BSC - Biological Safety Cabinet CD3 - Cluster of differentiation 3; a surface marker protein of T lymphocytes CF - centrifugal force Complete medium for CM1-TIL, #1 Complete medium for CM2-TILs, # CMO - Contract Manufacturing Organization CO2 - Carbon dioxide EtOH - Ethyl alcohol GMP - Standards for pharmaceutical manufacturing management and quality control Gy-Gray IL-2 - Interleukin 2 ·IU - International Unit LN2 - Liquid Nitrogen MiniREP - Mini Rapid Expansion Protocol ml - milliliters ·MNC-mononuclear cells NA - Not Applicable OKT3-MACS GMP CD3 pure (clone OKT3) antibody PPE - Personal Protective Equipment Pre-REP - Before the rapid expansion protocol QS - quantity; fill to this amount REP - Rapid Expansion Protocol TIL - tumor infiltrating lymphocytes T25-25cm2 tissue culture flask μg - microgram μl - microliter

[0379] Equipment, software, and materials

[0375] Device ·BSC (Biological Safety Cabinet) Liquid nitrogen freezer Temperature-controlled water bath ·Centrifuge with swinging bucket rotor Humidified tissue culture incubator Pipet Aid 2~20μl pipettor 20~200μl pipettor 100-1000μl pipettor Automated cell counter

[0380]

[0376] Material 15ml conical centrifuge tube, sterile 50ml conical centrifuge tube, sterile CM1 CM2 ·AIM V Medium CTS (therapeutic grade) Cell counterstaining solution IL-2 MACS GMP CD3 pure (clone OKT3) antibody Sterile disposable serum pipettes Sterile disposable volumetric pipettes Sterile pipette tips 24-well tissue culture plates T25 flask (Greiner #690175) 5.3.14. Zippered Storage Bags

[0381] procedure background

[0377] The REP (Step D) of TIL expansion culture included γ-irradiated, growth-arrested TILs. MNC feeder cells were required. TILs were stimulated to expand in the REP (Step D) flasks by binding membrane receptors on the feeder MNCs with anti-CD3 (clone OKT3) antibodies, which crosslinked the TILs. Feeder lots were prepared from leukapheresis of whole blood collected from individual donors. The leukapheresis product was centrifuged through Ficoll-Hypaque, washed, irradiated, and stored frozen under GMP conditions.

[0382]

[0378] Patients receiving TIL therapy should be aware of the risk of graft-versus-host disease (GVHD). It was important that the subjects were not injected with live feeder cells, which therefore halted proliferation by gamma-irradiating the cells, resulting in double-stranded DNA breaks that caused the MNC cells to lose viability upon re-culture.

[0383] Criteria and Experimental Setup Feeder lots were judged on two criteria: 1) TI in co-culture its ability to expand L >100-fold, and 2) its inability to replicate.

[0384] Two primary pre-REP TILs grown in upright T25 tissue culture flasks Feeder lots were tested in a mini-REP format utilizing the following lines: Because each TIL line was unique in its ability to proliferate in response to activation in the REP, feeder lots were tested against two different TIL lines. As a control, lots of irradiated MNC feeder cells historically shown to meet criteria 1) and 2): (1) their ability to expand TILs >100-fold in coculture, and (2) their inability to replicate were run in parallel with the test lots.

[0385] Ensuring that all lots tested in a single experiment receive equivalent testing Therefore, a sufficient stock of the same pre-REP TIL line was used for testing all conditions and all feeder lots. There were a total of six T25 flasks for each lot of feeder cells tested: Pre-REP TIL strain #1 (2 flasks) Pre-REP TIL strain #2 (2 flasks) Feeder control (2 flasks) Note: Flasks containing TIL lines #1 and #2 determined the ability of the feeder lot to expand TILs. Feeder control flasks determined the inability of the feeder lot to replicate.

[0386] Experimental protocol Day -2 / 3, thawing of TIL stocks CM2 medium was prepared according to Example 5, Pre-REP and REP medium preparation. CM2 was warmed in a 7°C water bath. 40 ml of CM2 supplemented with 3000 IU / ml IL-2 was prepared and kept warm until use. 20 ml of pre-warmed CM2 without IL-2 was placed in each of two 50 ml conical tubes labeled with the name of the TIL line used. Two designated pre-REP TIL lines were removed from LN2 storage, and the vials were transferred to the tissue culture room. The TIL line identification form was recorded. Vials were thawed by placing them inside a sealed zippered storage bag in a 37°C water bath until only a small amount of ice remained. The thawed vials were sprayed or wiped with 70% ethanol, and the vials were transferred to the BSC. Using a sterile transfer pipette, the contents of the vial were immediately transferred into 20 ml of CM2 in the labeled, prepared 50 ml conical tube. Cells were washed by QS (fill to this volume) to 40 ml with CM2 without IL-2. The cells were centrifuged at 400×CF for 5 minutes. The supernatant was aspirated and resuspended in 5 ml of warm CM2 supplemented with 3000 IU / ml IL-2. Small aliquots (20 μl) were removed in duplicate and the cells were counted using an automated cell counter. The counts were recorded. While counting, the 50 ml conical tube containing the TIL cells was placed in a humidified 37°C, 5% CO2 incubator with the cap loosened to allow gas exchange. The cell concentration was determined, and the TILs were cultured at a concentration of 1×10 in CM2 supplemented with 3000 IU / ml IL-2. 6 The TILs were diluted to 1000 cells / ml and cultured in 2 ml per well of a 24-well tissue culture plate in a humidified 37°C incubator in as many wells as needed until day 0 of the mini-REP. To avoid confusion and potential cross-contamination, different TIL lines were cultured in separate 24-well tissue culture plates.

[0387] Day 0, Start Mini-Rep Prepare sufficient CM2 medium for the number of feeder lots to be tested (e.g., 1 (For testing four feeder lots at a time, 800 ml of CM2 medium is prepared.) A portion of the CM2 prepared in Example 5 was divided into aliquots, to which 3000 IU / ml IL-2 was added for culturing the cells (e.g., for testing four feeder lots at a time, 500 ml of CM2 medium containing 3000 IU / ml IL-2 is prepared). The remaining CM2 without IL-2 was used to wash the cells as described below.

[0388] TILs were prepared 7.3.2.4. Working with each TIL line separately to prevent cross-contamination, the 24-well plates containing the TIL cultures were removed from the incubator and transferred to the BSC. 7.3.2.5. Using a sterile transfer pipette or a 100-1000 μl pipettor and tips, remove approximately 1 ml of media from each well of working TILs and place into an unused well of a 24-well tissue culture plate. This will be used to wash wells. 7.3.2.6. A fresh, sterile volumetric pipette or 100-1000 μl pipetter and Using a tip, the remaining medium was mixed with the TILs in the well to resuspend the cells, and the cell suspension was then transferred to a 50 ml conical tube labeled with the TIL name, and the volume was recorded. 7.3.2.7. The wells were washed with the reserved medium and the volume was transferred to the same 50 ml conical tube. 7.3.2.8. The cells were spun at 400x CF and the cell pellet was collected. 7.3.2.9. The medium supernatant was aspirated off and the cell pellet was resuspended in 2-5 ml of CM2 medium containing 3000 IU / ml IL-2; the volume used was based on the number of wells harvested and the size of the pellet - a volume >1.3 x 10 6 was sufficient to ensure a concentration of cells / ml. 7.3.2.10. Using a serological pipette, the cell suspension was mixed thoroughly and the volume recorded. 7.3.2.11. 200 μl was removed and the cells were counted using an automated cell counter. 7.3.2.12. While counting, the 50 ml conical tube containing the TIL cells was placed in a humidified 5% CO2, 37°C incubator with the cap loosened to allow gas exchange. 7.3.2.13. The count was recorded. 7.3.2.14. Remove the 50 ml conical tube containing the TIL cells from the incubator and soak the cells at 1.3 x 10 in warm CM2 supplemented with 3000 IU / ml IL-2. 6 The cells were resuspended at a concentration of 1000 cells / ml. The 50 ml conical tube was placed back into the incubator with the cap loosened. 7.3.2.15 If desired, the original 24-well plate was left intact for re-culture of the remaining TILs. 7.3.2.16. Repeat steps 7.3.2.4 through 7.3.2.15 for the second TIL line. 7.3.2.17. Immediately prior to plating TILs into experimental T25 flasks, plate 1.3 x 10 TILs by following step 7.3.2.35 below. 5 Diluted 1:10 to a final concentration of cells / ml.

[0389] Prepare MACS GMP CD3 pure (OKT3) working solution 7.3.2. Remove the OKT3 stock solution (1 mg / ml) from the 18.4°C refrigerator and place it in the BSC. 7.3.2.19. A final concentration of 30 ng / ml OKT3 was used in the miniREP medium. 7.3.2.20. 600 ng of OKT3 was required per 20 ml in each T25 flask in the experiment; this is the equivalent of 60 μl of a 10 μg / ml solution per 20 ml, or 360 μl for all six flasks tested for each feeder lot. For each feeder lot to be tested, make 400 μl of a 1:100 dilution of 1 mg / ml OKT3 for a working concentration of 10 μg / ml (e.g., to test 4 feeder lots at a time, make 1600 μl of a 1:100 dilution of 1 mg / ml OKT3: 16 μl of 1 mg / ml OKT3 + 1.584 ml of CM2 medium containing 3000 IU / ml IL-2).

[0390] Prepare a T25 flask 7.3.2.22. Each flask was labeled with the name of the TIL line tested, the flask replicate number, the feeder lot number, the date, and the analyst's initials. 7.3.2.23. Flasks were filled with CM2 medium before preparing the feeder cells. 7.3.2.24. Place flasks in a humidified 5% CO2 incubator at 37°C to keep the medium warm until the remaining components were added. 7.3.2.25. After preparing the feeder cells, follow the instructions in Table 14, Flask Setup, below. Components were added to the CM2 in each flask as indicated in the setup.

[0391] [Table 13]

[0392] Feeder cells were prepared 7.3.2.26 For this protocol, a minimum of 78 x 10 per lot tested 6 Each 1 ml vial frozen by SDBB ​​contained 100 x 10 feeder cells at the time of freezing. 6 Assuming a 50% recovery rate upon thawing from LN2 storage, each REP contained an estimated 100 x 10 viable cells. 6 Given viable cells, it was recommended to thaw at least two 1 ml vials of feeder cells per lot, or if supplied in 1.8 ml vials, only one vial would provide sufficient feeder cells. 7.3.2.27. Prior to thawing the feeder cells, pre-warm approximately 50 ml of IL-2-free CM2 for each feeder lot to be tested. 7.3.2.28. The designated feeder lot vials were removed from LN2 storage, placed in zippered storage bags, and placed on ice. The vials were transferred to the tissue culture room. 7.3.2.29. The vials were thawed in the closed zipper storage bag by immersion in a 37°C water bath. 7.3.2.30. Remove the vial from the zip-top bag, spray or wipe with 70% EtOH, and transfer the vial to the BSC. Using a transfer pipette, the contents of the feeder vial were immediately transferred into 30 ml of warm CM2 in a 50 ml conical tube. The vial was washed with a small amount of CM2 to remove any remaining cells in the vial. 7.3.2.3 Centrifuge at 2400×CF for 5 minutes. 7.3.2.33. The supernatant was aspirated and resuspended in 4 ml warm CM2 + 3000 IU / ml IL-2. 7.3.2.34. 200 μl was removed and the cells were counted using an automated cell counter. The count was recorded. 7.3.2.35 Cells were cultured at 1.3 x 10 in warm CM2 + 3000 IU / ml IL-2. 7 cells / ml.

[0393] Setting up the co-culture 7.3.2.36 TIL cells 1.3 x 10 6 cells / ml~1.3×10 5 Each TIL line was worked independently to prevent cross-contamination. 7.3.2.36.1. Add 4.5 ml of CM2 medium to a 15 ml conical tube. Ta. 7.3.2.36.2. Remove the TIL cells from the incubator and resuspend thoroughly using a 10 ml serological pipette. 7.3.2.36.3.1.3×10 60.5 ml of cells from the cells / ml TIL suspension was removed and added to 4.5 ml of media in a 15 ml conical tube. The TIL stock vial was returned to the incubator. 7.3.2.36.4. Mix thoroughly. 7.3.2.36.5. Repeat steps 7.3.2.36.1 through 7.3.2.36.4 for the second TIL line. 7.3.2.36.6. If more than one feeder lot was tested at a time, the TILs were diluted to a lower concentration for each feeder lot immediately prior to plating the TILs. 7.3.2.37. Flasks containing pre-warmed medium for a single feeder lot were transferred from the incubator to the BSC. 7.3.2.38. Mix the feeder cells by pipetting up and down several times with a 1 ml pipette tip and add 1 ml (1.3 x 10 7 cells) were transferred to each flask of the feeder lot. 7.3.2.39. To each flask was added 60 μl of OKT3 working stock (10 μg / ml). 7.3.2.40. The two control flasks were returned to the incubator. 7.3.2.41.1 ml (1.3 x 10 5 ) each TIL lot was transferred to the corresponding indicated T25 flask. The flask was returned to the incubator and incubated upright, undisturbed until day 5. 7.3.2.43. Repeat 7.3.2.36 through 7.3.2.42 for all feeder lots tested.

[0394] On day 7.3.3.5, the medium was changed. 7.3.3.1. Prepare CM2 containing 3000 IU / ml IL-2. 10 ml is required for each flask. 7.3.3.2 To prevent cross-contamination, flasks from a single feeder lot were handled at a time. Flasks were removed from the incubator and transferred to the BSC, and care was taken not to disturb the cell layer at the bottom of the flask. 7.3.3.3. 10 ml of medium was gently removed from the flask and discarded. 7.3.3.4. Repeat for all flasks, including the control flask. 7.3.3.5. Using a 10 ml pipette, 10 ml of warm CM2 containing 3000 IU / ml IL-2 was transferred to each flask. 7.3.3.6. The flasks were returned to the incubator and incubated upright for up to 7 days. 7.3.3.7. Repeat steps 7.3.3.1 through 7.3.3.6 for all feeder lots tested.

[0395] 7.3.4.7th day, collect 7.3.4.1. To prevent cross-contamination, flasks from a single feeder lot were handled at a time. 7.3.4.2. Remove the flask from the incubator and transfer it to the BSC, taking care not to disturb the cell layer at the bottom of the flask. 7.3.4.3. Without disturbing the cells growing on the bottom of the flask, 10 ml of medium was removed from each test flask and 15 ml of medium from each of the control flasks. 7.3.4.4. Using a 10 ml serological pipette, resuspend the cells in the remaining medium and mix thoroughly to break up any cell clumps. 7.3.4.5. On day 7, the volume of each flask was recorded. 7.3.4.6. After thoroughly mixing the cell suspension by pipetting, 200 μl was removed and the cells were counted. 7.3.4.7. TILs were counted using appropriate standard procedures in conjunction with an automated cell counter device. Counts were recorded on days 7, 3, 4, 8, and 7. 7.3.4.9. Repeat steps 7.3.4.1 through 7.3.4.8 for all feeder lots tested. 7.3.4.10. Feeder control flasks were assessed for replication incompetence and flasks containing TILs were assessed for fold expansion from day 0 according to the criteria listed in Figure 2.

[0396] 7.3.5. Day 7, continuation of feeder control flasks through day 14 7.3.5.1. After the feeder control flasks were counted on day 7, 15 ml of fresh CM2 medium containing 3000 IU / ml IL-2 was added to each control flask. 7.3.5.2. The control flask was returned to the incubator and incubated in an upright position until day 14.

[0397] 7.3.6. Day 14, Prolonged Non-Growth in Feeder Control Flasks 7.3.6.1 To prevent cross-contamination, flasks from a single feeder lot were handled at a time. 7.3.6.2 Remove the flask from the incubator and transfer it to the BSC, taking care not to disturb the cell layer at the bottom of the flask. 7.3.6.3. Approximately 17 ml of medium was removed from each control flask without disturbing the cells growing on the bottom of the flask. 7.3.6.4. Using a 5 ml serological pipette, resuspend the cells in the remaining medium and mix thoroughly to break up any cell clumps. 7.3.6.5. The volume of each flask was recorded. 7.3.6.6. After thoroughly mixing the cell suspension by pipetting, 200 μl was removed and the cells were counted. 7.3.6.7. TILs were counted using appropriate standard procedures in conjunction with an automated cell counter device. Counts were recorded on days 7, 3, 6, 8 and 14. 7.3.6.9. Repeat steps 7.3.4.1 through 7.3.4.8 for all feeder lots tested.

[0398] Expected Results and Eligibility Criteria Expected Results

[0384] The dose of gamma irradiation was sufficient to render the feeder cells replication-incompetent. All lots were expected to meet the acceptance criteria and also demonstrated a reduction in the total number of viable feeder cells remaining at day 7 of REP culture compared to day 0.

[0399] All feeder lots achieved 100% TIL growth by day 7 of REP culture. It was expected to meet the criteria of 2x magnification.

[0400]

[0386] Day 14 counts of the feeder control flasks were significantly higher than the non-growth seen on day 7. The trend was expected to continue.

[0401] Compliance criteria Each replicate TIL line tested for each lot of feeder cells was: The following eligibility criteria had to be met:

[0402] The match was twofold as follows (outlined in Figure 2 , match criteria): In the presence of 30 ng / ml OKT3 antibody and 3000 IU / ml IL-2 We determined whether the radiation dose was sufficient to render MNC feeder cells replication-incompetent when cultured at RT.

[0403] Replication failure was determined by automated cell counting on days 7 and 14 of REP. The total viable cell count (TVC) was determined at the time of the assay.

[0404] The eligibility criteria was "no growth," which was determined by the number of cells cultured on day 0 of the REP. This means that the total number of viable cells did not increase from the initial number of viable cells on days 7 and 14.

[0405] Determine the ability of feeder cells to support TIL expansion

[0391] Viable cell expansion fold points from the start of culture on day 0 of REP to day 7 of REP TIL growth was measured by

[0406] On day 7, TIL cultures had at least A 100-fold expansion (ie, greater than 100 times the total number of viable TIL cells that initiated the culture on REP day 0) was achieved.

[0407] MNC feeder lots that did not meet these two criteria above were typically was essentially excluded.

[0408]

[0394] The compatibility criteria are met, but as judged by one skilled in the art, the same pre-REP TIL line Any MNC feeder lot deemed to perform poorly in terms of TIL expansion capacity compared to previous feeder lots tested in parallel may have been excluded (see Table 15 below for eligibility criteria used).

[0409] [Table 14]

[0410] In the presence of 30 ng / ml OKT3 antibody and 3000 IU / ml IL-2 We determined whether the radiation dose was sufficient to render MNC feeder cells replication-incompetent when cultured at RT. 10.2.2.1.1 Replication incompetence was determined by total viable cell count (TVC) as determined by automated cell counting on days 7 and 14 of the REP. 10.2.2.1.2 The eligibility criterion was "no growth," meaning that there was no increase in total viable cell count on days 7 and 14 from the initial viable cell count at the start of culture on day 0 of the REP. 10.2.2.2 The ability of feeder cells to support TIL expansion was determined. 10.2.2.2.1 TIL growth was measured in terms of fold expansion of viable cells from the initiation of culture on day 0 of the REP to day 7 of the REP. 10.2.2.2.1 On Day 7, TIL cultures achieved a minimum of 100-fold expansion (i.e., greater than 100-fold the total viable TIL cell number that initiated the culture on REP Day 0) as determined by automated cell counting. 10.2.2.3 If a lot failed to meet the above two criteria, the lot was retested in accordance with the contingency plan outlined in Section 10.3 below. 10.2.2.4 After retesting of non-conforming lots, any MNC feeder lot that did not meet the two conformance criteria in both the original adjudication and the contingency testing was excluded. 10.2.2.5 Any MNC feeder lot that met the eligibility criteria but was determined to perform poorly in terms of TIL expansion capacity compared to previous feeder lots tested in parallel with the same pre-REP TIL line was appropriately excluded.

[0411]

[0396] Contingency Test for MNC Feeder Lots that Do Not Meet the Eligibility Criteria to 10.3.1 If an MNC feeder lot met any of the eligibility criteria outlined in Section 10.2 above, the lot was retested using the following steps to rule out simple operator error as a cause. 10.3.2 If two or more satellite test vials remain in a lot, If a lot had one or no satellite test vials remaining, the lot was rejected based on the eligibility criteria listed in Section 10.2 above. 10.3.3 Two trained personnel, including the person who originally judged the lot in question, must both test the lot at the same time. 10.3.4 Sections 7.2-7.3 were repeated and the lot in question was re-determined. 10.3.5 Each person could have tested the subject lot as well as the control lot (as defined in Section 7.2.4 above). 10.3.6 To be qualified, the subject lot and the control lot had to achieve the conformance criteria in Section 10.2 for both personnel performing the contingency testing. 10.3.7 Once these criteria were met, the lot could then be released for CMO use as outlined in Section 10.2 above.

[0412] Example 7: Procedure for qualifying individual lots of gamma-irradiated peripheral blood mononuclear cells This example demonstrates that individual lots of gamma-irradiated peripheral blood mononuclear cells (PBMCs) are This paper describes a novel, simplified procedure for qualifying irradiated PBMC cell lots for use as allogeneic feeder cells in the exemplary method described in [1]. This example provides a protocol for qualifying irradiated PBMC cell lots for use in producing clinical TIL lots. Each irradiated PBMC lot was prepared from a separate donor. Through over 100 qualification protocols, it has been shown in each case that irradiated PBMC lots from SDBB ​​(San Diego Blood Bank) can expand TILs >100-fold on day 7 of REP. This improved qualification protocol is intended to apply to irradiated donor PBMC lots from SDBB ​​that must still be tested to confirm that the gamma radiation dose they received was sufficient to render them replication-incompetent. A donor PBMC lot was deemed "qualified" for use in producing clinical TIL lots if it was demonstrated to remain replication-incompetent for 14 days.

[0413] Keywords and Definitions μg - microgram μl - microliter AIM-V - Commercial Cell Culture Media, Biological Safety Cabinet BSC-differentiation cluster Complete medium #2 for CD-TILs CM2 - CM2 supplemented with 3000 IU / ml IL-2 CM2IL2 - Pharmaceutical Contract Manufacturing Organization CO2 - Carbon dioxide EtOH - Ethanol GMP - Standards for pharmaceutical manufacturing management and quality control Gy-Gray IL-Interleukin IU - International Unit LN2 - Liquid Nitrogen MI - milliliters NA - Not Applicable OKT3 - Anti-CD3 monoclonal antibody name P20 - 2~20μl pipettor P200 - 20~200μl Pipettor PBMC-peripheral blood mononuclear cells P1000 - 100~1000μl Pipettor PPE - Personal Protective Equipment REP - Rapid Expansion Protocol SDBB-San Diego Blood Bank TIL - tumor-infiltrating lymphocytes T25-25cm2 tissue culture flask ×g - "~ times gravity" - a measure of relative centrifugal force

[0414]

[0399] Specimens included irradiated donor PBMCs (SDBB). procedure background 7.1.1 The current standard REP of TILs required γ-irradiated, growth-arrested PBMCs. In culture, membrane receptors on PBMCs bind to anti-CD3 (clone OKT3) antibodies, crosslinking TILs and stimulating their expansion. PBMC lots were prepared from leukapheresis of whole blood collected from individual donors. The leukapheresis product was centrifuged through Ficoll-Hypaque, washed, irradiated, and stored frozen under GMP conditions. It is important that patients receiving TIL therapy are not infused with live PBMCs, as this can lead to graft-versus-host disease (GVHD). Therefore, donor PBMCs were subjected to gamma irradiation, which resulted in double-stranded DNA breaks that caused the PBMCs to lose viability upon re-culture, thereby arresting their proliferation. Judgment criteria 7.2.1 The criterion for the irradiated PBMC lots was their inability to replicate. Experimental setup 7.3.1 Feeder lots were tested in a mini-REP format as if co-cultured with TILs using upright T25 tissue culture flasks. 7.3.1.1 Control Lot: One lot of irradiated PBMCs, historically shown to meet the criteria in 7.2.1, was run in parallel with the experimental lot as a control. 7.3.2 Duplicate flasks were run for each lot of irradiated donor PBMC tested.

[0415] Experimental protocol All tissue culture work in this protocol was performed using aseptic technique in a BSC. And went. Day 0 7.4.1 Approximately 90 ml of CM2 medium was prepared for each lot of donor PBMC to be tested. The CM2 was kept warm in a 37°C water bath. 7.4.2 6×10 6 An aliquot of IU / ml IL-2 was thawed. 7.4.3 The CM2 medium was returned to the BSC, wiped down with 70% EtOH, and placed in the hood. For each lot of PBMC to be tested, approximately 60 ml of CM2 was removed into a separate sterile bottle. 6 x 10 thawed 6 IL-2 from a 1U / ml stock solution was added to this medium to a final concentration of 3000 IU / ml. The bottle was labeled "CM2 / IL2" (or similar) to distinguish it from unsupplemented CM2. 7.4.4 For each lot of PBMC to be tested, two T25 flasks were labeled. The labeling included, as a minimum: 7.4.4.1 Lot Number 7.4.4.2 Flask number (1 or 2) 7.4.4.3 Culture start date (day 0) OKT3 was prepared 7.4.5 Remove the anti-CD3 (OKT3) stock solution from the 4°C refrigerator and place it in the BSC. 7.4.6 A final concentration of 30 ng / ml OKT3 was used in the mini-REP medium. 7.4.7 Prepare a 10 μg / ml working solution of anti-CD3 (OKT3) from a 1 mg / ml stock solution. Store in the refrigerator until needed. 7.4.7.1 For each PBMC lot to be tested, 150 μl of a 1:100 dilution of anti-CD3 (OKT3) stock was prepared. For example, to test four PBMC lots at a time, 600 μl of 10 μg / ml anti-CD3 (OKT3) was prepared by adding 6 μl of a 1 mg / ml stock solution to 594 μl of CM2 supplemented with 3000 IU / ml IL-2. Flasks were prepared 7.4.8 19 ml of CM2 / IL-2 was added per flask to the labeled T25 flasks and the flasks were placed in a humidified 5% CO2 incubator at 37°C while the cells were prepared. Irradiated PBMCs were prepared 7.4.9 Each donor PBMC lot was handled separately to prevent potential cross-contamination of lots. 7.4.10 Vials of the PBMC lot to be tested were removed from LN2 storage and placed at -80°C or kept on dry ice until thawed. 7.4.11 For each lot to be thawed, 30 ml of CM2 (without IL-2) was placed into a 50 ml conical tube. Each tube was labeled with the different lot number of PBMCs to be thawed. The tubes were tightly capped and placed in a 37°C water bath until ready for use. If needed, the 50 ml conical tubes were returned to the BSC, wiped down with 70% EtOH, and placed in the hood. 7.4.12 Remove the vials of PBMC from cryogenic storage and place them in a floating tube rack in a 37°C water bath to thaw. Thawing was allowed to proceed until a small amount of ice remained in the vial. 7.4.13 Thawed vials were sprayed or wiped with 70% EtOH and transferred to the BSC. 7.4.14 Using a sterile transfer pipette, the contents of the vial were immediately transferred into 30 ml of CM2 in a 50 ml conical tube. Approximately 1 ml of medium was removed from the tube. The vial was rinsed with PBS; the rinse was poured back into the 50 ml conical tube. The cap was tightly closed and gently swirled to wash the cells. 7.4.15 Centrifuge at 400 x g for 5 minutes at room temperature. 7.4.16 The supernatant was aspirated and the cell pellet resuspended in 1 ml of warm CM2 / IL-2 using a 1000 μl pipette tip. Alternatively, the cell pellet was resuspended by dragging the capped tube along an empty tube rack before adding media. After resuspension of the cell pellet, the volume was brought to 4 ml using CM2 / IL-2 media. The volume was recorded. 7.4.17 A small aliquot (e.g., 100 μl) was removed and the cells counted using an automated cell counter. 7.4.17.1 Counts were performed in duplicate according to detailed automated cell counter SOPs. In many cases, it was necessary to perform a dilution of the PBMCs before performing the cell count. The recommended starting dilution was 1:10, but this could vary depending on the type of cell counter used. 7.4.17.2 Counts were recorded. 7.4.18 Increase the concentration of PBMCs to 1.3 x 10 using CM2 / IL-2 medium according to step 7.4.15.2 7 Adjust to cells / ml. Mix thoroughly by gently swirling or by gently aspirating up and down using a serological pipette. Culture flasks were set up 7.4.19 The two labeled T25 flasks were returned from the tissue culture incubator to the BSC. 7.4.20 The 10 μg / ml vial of anti-CD3 / OKT3 was returned to the BSC. 7.4.21 Add 1 ml of 1.3 x 10 7 The PBMC cell suspension was added. 7.4.22 To each flask was added 60 μl of 10 μg / ml anti-CD3 / OKT3. 7.4.23 The capped flasks were returned to the tissue culture incubator and allowed to grow undisturbed for 14 days. 7.4.24 The anti-CD3 / OKT3 vials were placed back in the refrigerator until needed for the next lot. 7.4.25 Repeat steps 7.4.9 to 7.4.24 for each lot of PBMC to be tested. Day 14: Measurement of non-proliferative activity of PBMCs 7.4.26 Working independently for each lot, duplicate T25 flasks were carefully returned to the BSC. 7.4.27 For each flask, a fresh 10 ml serological pipette was used to remove approximately 17 ml from each of the flasks, then the remaining medium was carefully drawn off to measure the volume remaining in the flask. The volume was recorded. 7.4.28 The sample was mixed thoroughly by pipetting up and down using the same serological pipette. 7.4.29 A 200 μl sample was removed from each flask for counting. 7.4.30 Cells were counted using an automated cell counter. 7.4.31 Repeat steps 7.4.26 to 7.4.31 for each lot of PBMC to be tested. Results and Eligibility Criteria result 10.1.1 The dose of gamma irradiation was sufficient to render the feeder cells replication incompetent. All lots are expected to meet the acceptance criteria and demonstrate a reduction in the total number of viable feeder cells remaining at day 14 of REP culture compared to day 0. Compliance criteria 10.2.1 For each irradiated donor PBMC lot tested, the following eligibility criteria were met: 10.2.2 "No growth" - means that the total viable cell count on day 14 was less than the initial viable cell count at the start of the culture on day 0 of the REP. 10.2.3 If a lot did not meet the above criteria, the lot was retested in accordance with the contingency testing procedures outlined in Section 10.4. 10.2.4 After retesting of non-conforming lots, any MNC feeder lots that did not meet the conformance criteria both in the original determination and in the contingency testing were excluded. Contingency testing of PBMC lots that did not meet eligibility criteria 10.4.1 If an irradiated donor PBMC lot did not meet the above eligibility criteria, the lot was retested using the following steps to rule out simple operator error as the cause of the non-compliance. 10.4.2 If two or more satellite vials remained in a lot, the lot was retested. If one or no satellite vials remained in a lot, the lot was rejected based on the compliance criteria in Section 10.2 above. 10.4.3 Whenever possible, two separate vials were tested independently by two trained personnel (preferably including the person who originally judged the lot in question). This was the preferred contingency testing method. In addition to separate vials of PBMC, both personnel could use the same reagents. 10.4.3.1. If two individuals were not available, one person tested two PBMC vials from the rejected lot, working independently on each vial. 10.4.4 Section 7.4, "Experimental Protocol," was repeated and the lot in question was re-characterized. 10.4.5 In addition to the problem lot, each contingency tester tested a control lot. 10.4.5.1 When two individuals perform contingency testing, both individuals independently tested the control lot. 10.4.5.2 If only one person is available to perform the contingency test, it is not necessary to run duplicates on the control lot. 10.4.5.3 To be eligible, a PBMC lot undergoing contingency testing must have replicates of both the control lot and the lot in question that meet the eligibility criteria in Section 10.2. 10.4.5.4 Once this criteria was met, the lot was then released for CMO use as outlined in Section 10.2.

[0416] Example 8: Comparison of pre- and post-cryopreservation TILs

[0401] Antibody citrate for samples and FMO controls before starting the sample preparation and staining procedure. These cocktails were stored in the dark at 4°C for up to 60 days. See the Cocktail Preparation section below.

[0417] [Table 15]

[0418] [Table 16]

[0419] [Table 17]

[0420] [Table 18]

[0421] [Table 19]

[0422] Compensation Control 1.1 drops of BD Comp beads were added to 11 tubes. 2. Tubes 1–7 were labeled with the chromophore from DF1 3. Tubes 8-10 were labeled with APCy7, BV421, and Ax647. 4. Tube 11 was for unlabeled beads. 5. 5 μL of antibody was added to each tube. 6. Incubate in the dark at room temperature for 10 to 30 minutes. Wash with 7.3 mL FACS buffer 8. Resuspended in 500uL 1% PFA. 9. Added one drop of BD Comp negative beads to each tube. 10. It was stored in the dark at 4°C and was usable for one week. Aqua Control: 1. One drop of Arc positive control was added to the tube labeled Aqua. 2. 3 μL of thawed aqua solution was added to the tube. 3. Steps 6-10 were repeated as above, except that negative Arc beads were used in step 9.

[0423] [Table 20]

[0424] Example 9: Tumor infiltrating lymphocytes (TILs) are remarkably stable in infusion phenotype after cryopreservation Summary Background:

[0402] This example demonstrates the ability of tumor-infiltrating lymphocytes (TILs) to be differentiated with the ultimate goal of developing a therapeutic TIL population. This paper discusses the development of tumor leukocyte (TIL)-based cancer immunotherapy. Cryopreservation of TILs reduces time constraints and allows for safe shipping of the final cell product. (Axelsson S, Faresjo M, Hedman M, Ludvigsson J, Casas R: Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children. Cryobiology 2008, 57:201-8)

[0425]

[0403] Here, fresh TIL samples and freeze / thawed TIL samples were analyzed for the expression of individual phenotypic markers. TIL samples were evaluated to assess whether phenotypic changes occur in cryopreserved TILs (see, e.g., Sadeghi A, Ullenhag G, Wagenius G, Toetterman TH, Eriksson F: Rapid expansion of T cells: Effects of culture and cryopreservation and the importance of short-term cell recovery. Acta Oncol. 2013,52:978-86).

[0426] result: Fresh TILs were compared with thawed TILs to show increased expression of CD4, CD8, NK, and TCRαβ. No significant differences were observed in activation or memory markers. The activation state of TILs, as defined by HLA-DR, CD38, and CD69 expression, was maintained, while the regulatory molecules LAG-3 and TIM-3 showed slightly decreased expression. Additionally, viability was greater than 86% for both fresh and thawed products.

[0427] method:

[0405] By culturing melanoma tumor fragments in IL-2 (6000 IU / ml) Got the pre-REP TIL.

[0428]

[0406] Irradiated allogeneic PBMC feeder cells were cultured in GREX-100 flasks with OKT3 and Rapid expansion protocol (REP) cells were initiated with IL-2 for 11-14 days.

[0429]

[0407] Cultured cells were cryopreserved in 5% DMSO.

[0430] Using a four-panel of lineage, differentiation, activation, and regulatory markers, Flow cytometric evaluation of fresh and thawed TILs was performed after 1-2 hours of incubation in IL-2.

[0431] Conclusion:

[0409] Except for minor changes in some regulatory molecules, cryopreservation does not measure the phenotypic characteristics of TILs. The inventors are investigating the feasibility of using cryopreserved TILs in a clinical setting.

[0432] Example 10: Memory cell subsets in fresh and ReREP TIL populations In the previous experiment, no central memory subset was observed in the fresh TIL population. (See Figure 8.) However, after ReREP, nearly 60% of central memory cells were recovered, as provided in Table 22 below.

[0433] Based on raw counts, resting cells have a slightly higher CD4 population than non-resting cells. As expected, there was a high proportion of CD8 cells overall. Among CD8 cells, there was an approximately 60 / 40 split of CM (central memory - Q3) / EM (effector memory - Q4). The CD8+CD28+ expression appears interesting; it is higher in resting cells. See also Figure 9 and Figures 10A-10B. See also Figure 15.

[0434] Example 11: Administration of autologous tumor-infiltrating lymphocytes (TILs) in melanoma patients

[0412] Administration of autologous tumor-infiltrating lymphocytes (TILs) in melanoma patients has been shown to be effective in 5 years at the NCI. 5% at Moffitt Cancer Center, 38% at MD Anderson Cancer Center The study showed an overall response rate of 48% at the MD Anderson Cancer Center and 40% at the Ella Cancer Institute, Sheba, Israel. The durable responses observed in melanoma patients using this therapy may enable broad application to other solid tumors. As shown herein, the feasibility of growing TILs and developing TIL therapy for other solid tumors is demonstrated. This example provides data demonstrating the successful expansion and characterization of tumor-infiltrating lymphocytes (TILs) from non-melanoma tumors. See Figures 12-14.

[0435]

[0413] Phenotypic characterization of TILs from bladder, cervical, and lung cancers has been reported. TILs from the head showed a variable distribution of CD8+ and CD4+ T cells. TILs expanded from TNBC were >80% CD4+ Most cultures, regardless of tumor, had less than 20% CD56+ NK cells.

[0436] TILs were prepared by: a. Wash the obtained tumor with HBSS; b. Dicing the tumor into fragments (e.g., 2-3 mm3 fragments); c. Place tumor fragments into G-REX 10 cell culture flasks containing medium containing serum and IL-2; d. Change the medium on day 7 and every 4-5 days from day 11 to day 21; and e. Cell count, viability assessment, and phenotyping followed by cryopreservation for further purposes, including but not limited to, further delivery to a patient for treatment of tumors as described herein.

[0437] As demonstrated herein, TILs are expressed in the lung, bladder, head and neck, cervix, and and grown from TNBC patient tumors.

[0438] Furthermore, as demonstrated herein, lung, bladder, and cervical tumors , showed a greater proportion of CD8+ TILs. Head and neck and TNBC tumors were predominantly CD4+ TILs. In addition, further characterization of CD4+ and CD8+ TILs demonstrated effector memory phenotype cells that were also CD27+ and CD28+.

[0439]

[0417] The examples provided above illustrate how embodiments of the compositions, systems, and methods of the present invention can be used. These and other references are provided so as to provide a complete disclosure and description to those skilled in the art of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Variations of the above-described modes for carrying out the invention that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0440]

[0418] All headings and section designations are for clarity and reference only. For example, those skilled in the art will appreciate the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention as described herein.

[0441]

[0419] All references cited herein are to each individual publication or patent or patent issue. This application is hereby incorporated by reference in its entirety and for all purposes to the same extent as if each such application were specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0442]

[0420] Many modifications and variations of this application may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

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

2. The method comprises:

10. The method of claim 1, further comprising: (iv) performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (iii), wherein the larger therapeutic TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population.

3. 3. The method of claim 2, wherein after step (iii), the cells are removed from the cell culture and cryopreserved in storage medium before performing step (iv).

4. 4. The method of claim 3, wherein the cells are thawed prior to performing step (iv).

5. 5. The method of any one of claims 1 to 4, wherein step (iv) is repeated 1 to 4 times to obtain sufficient TILs to provide a therapeutically effective dosage of TILs in the therapeutic TIL population.

6. 6. The method of any one of claims 1 to 5, wherein steps (i) to (iii) or (iv) are carried out within a period of about 40 days to about 50 days.

7. 7. The method of any one of claims 1 to 6, wherein steps (i) to (iii) or (iv) are carried out within a period of about 42 days to about 48 days.

8. 8. The method of any one of claims 1 to 7, wherein steps (i) to (iii) or (iv) are carried out within a period of about 42 days to about 45 days.

9. 9. The method of any one of claims 1 to 8, wherein steps (i) to (iii) or (iv) are carried out within about 44 days.

10. 10. The method of any one of claims 1 to 9, wherein the cells from step (iii) or (iv) express CD4, CD8, and TCRαβ to the same extent as freshly harvested cells.

11. The method of claim 1 , wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

12. The method of claim 11, wherein the PBMCs are added to the cell culture in step (iii) on any day from 9 to 17.

13. 13. The method of any one of claims 2 to 12, wherein the effector T cells and / or central memory T cells in the therapeutic TIL population of step (iv) exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to effector T cells and / or central memory T cells in the third cell population.

14. 14. The method of claim 13, wherein the effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

15. The method of any one of claims 1 to 14, wherein the APC is an artificial APC (aAPC).

16. 16. The method of any one of claims 1 to 15, further comprising transducing the first TIL population with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

17. 17. The method of any one of claims 1 to 16, further comprising transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

18. The method of any one of claims 1 to 17, wherein the therapeutic TIL population is infused into a patient.

19. 10. The method of claim 1, wherein step (iii) further comprises removing the cells from the cell culture medium.

20. 10. The method of claim 1, wherein step (iii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dosage of TILs in the therapeutic TIL population.

21. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ The method of claim 20,

22. 10. An expanded TIL population produced by the method of claim 1.

23. 10. An expanded TIL population produced by the method of claim 1, wherein the expanded TILs have at least a two-fold increase in basal glycolysis when compared to thawed cryopreserved TILs.

24. A method for assessing the metabolic activity of a TIL cell population generated by the method of claim 1, comprising measuring the basal glycolysis of the cells.

25. A method for assessing the metabolic activity of a TIL cell population generated by the method of claim 1, comprising measuring the basal respiration of the cells.

26. A method for assessing the metabolic activity of a TIL cell population generated by the method of claim 1, comprising measuring the spare respiratory capacity (SRC) of the cells.

27. A method for assessing the metabolic activity of a TIL cell population generated by the method of claim 1, comprising measuring the glycolytic reserve of the cells.

28. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (i) performing a first expansion culture by culturing a first population of TILs from a tumor resected from a patient in a cell culture medium containing IL-2 to obtain a second population of TILs; and (ii) performing a second expansion culture by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to obtain a third TIL population, wherein the third TIL population is at least 100-fold more numerous than the second TIL population, and wherein the second expansion culture is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population comprising increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population.

29. The method comprises:

29. The method of claim 28, further comprising: (iii) performing an additional second expansion of the third TIL population by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (ii), wherein the larger therapeutic TIL population exhibits increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population.

30. 30. The method of claim 29, wherein the cells from the cell culture medium of step (ii) are removed and cryopreserved in a storage medium prior to step (iii).

31. 31. The method of claim 30, wherein the cells are thawed prior to step (iii).

32. 29. The method of claim 28, wherein step (ii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dosage of TILs in the therapeutic TIL population.

33. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ 33. The method of claim 32, wherein

34. The method of any one of claims 28 to 33, wherein the APCs are peripheral blood mononuclear cells (PBMCs).

35. 35. The method of any one of claims 29 to 34, wherein the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

36. 36. The method of claim 35, wherein the effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

37. 1. A method of treating a subject with cancer comprising administering expanded tumor infiltrating lymphocytes (TILs), (i) obtaining a first population of TILs from a tumor resected from a patient; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; (iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 100-fold more numerous than the second TIL population, and the second expansion is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population that includes increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population; and (iv) administering a therapeutically effective dosage of said third population of TILs to said patient. A method comprising:

38. 38. The method of claim 37, wherein the method further comprises, prior to step (iv), performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (iii), wherein the larger therapeutic TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population.

39. 38. The method of claim 37, wherein after step (ii), the cells are removed from the cell culture medium and cryopreserved in storage medium prior to the additional second expansion culture of claim 38.

40. 40. The method of claim 39, wherein the cells are thawed prior to the additional second expansion culture of claim 38.

41. 38. The method of claim 37, wherein step (iii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dosage of TILs in the therapeutic TIL population.

42. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ 42. The method of claim 41 , wherein

43. The method of any one of claims 37 to 42, wherein the APCs are peripheral blood mononuclear cells (PBMCs).

44. 44. The method of any one of claims 37 to 43, wherein the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

45. 45. The method of claim 44, wherein the effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

46. 46. ​​The method of any one of claims 37 to 45, wherein the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer.

47. 1. A method of treating a subject with cancer comprising administering expanded tumor infiltrating lymphocytes (TILs), (i) A first collection of TILs from tumors resected from patients in cell culture medium containing IL-2. performing a first expansion by culturing the group to obtain a second population of TILs; (ii) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to obtain a third TIL population, wherein the third TIL population is at least 100-fold more numerous than the second TIL population, and the second expansion is performed for at least 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population that includes increased effector T cell and / or central memory T cell subpopulations compared to the second TIL population; and (iii) administering a therapeutically effective dosage of said therapeutic TIL population to said patient. A method comprising:

48. 48. The method of claim 47, wherein the method further comprises, prior to step (iii), performing an additional second expansion culture by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion culture is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (ii), wherein the larger therapeutic TIL population comprises increased effector T cell and / or central memory T cell subpopulations compared to the third TIL population.

49. 49. The method of claim 47, wherein the cells from the cell culture medium of step (ii) are removed and cryopreserved in storage medium prior to the additional second expansion culture of claim 48.

50. 50. The method of claim 49, wherein the cells are thawed prior to the additional second expansion culture of claim 48.

51. 48. The method of claim 47, wherein step (ii) is repeated 1 to 4 times to obtain enough TILs to provide a therapeutically effective dosage of TILs in the therapeutic TIL population.

52. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ 52. The method of claim 51 , wherein

53. The method of any one of claims 47 to 52, wherein the APCs are peripheral blood mononuclear cells (PBMCs).

54. 54. The method of any one of claims 47 to 53, wherein the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to the effector T cells and / or central memory T cells in the third cell population.

55. 55. The method of claim 54, wherein the effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

56. 56. The method of any one of claims 47 to 55, wherein the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer.

57. 1. A method for assaying TILs, comprising: (i) obtaining a first population of TILs; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; and (iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population to generate a third TIL population, wherein the third TIL population is at least 50-fold more numerous than the second TIL population; (iv) harvesting, washing, and cryopreserving the third population of TILs; (v) storing the cryopreserved TILs at cryogenic temperatures; (vi) thawing the third TIL population to provide a thawed third TIL population; and (vii) performing an additional second expansion of a portion of the thawed third TIL population for a reREP period of at least 3 days by adding IL-2, OKT-3, and APC to the cell culture medium of the third population, wherein the third expansion is performed to obtain a fourth TIL population, and comparing the number of TILs in the fourth TIL population with the number of TILs in the third TIL population to determine a ratio; (viii) determining whether the thawed TIL population is suitable for administration to a patient based on the ratio of step (vii); (ix) administering to the patient a therapeutically effective dose of the thawed third TIL population when the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is determined in step (viii) to be greater than 5:

1. A method comprising:

58. 58. The method of claim 57, wherein the reREP period is performed until the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is greater than 50:

1.

59. The number of TILs sufficient for a therapeutically effective dose is approximately 2.3 x 10 １０ ~Approx. 13.7×10 １０ 58. The method of claim 57, wherein

60. 60. The method of any one of claims 57 to 59, wherein steps (i) to (vii) are carried out within a period of about 40 days to about 50 days.

61. 60. The method of any one of claims 57 to 59, wherein steps (i) through (vii) are carried out within a period of about 42 days to about 48 days.

62. 60. The method of any one of claims 57 to 59, wherein steps (i) through (vii) are carried out within a period of about 42 days to about 45 days.

63. 60. The method of any one of claims 57 to 59, wherein steps (i) to (vii) are carried out within about 44 days.

64. 64. The method of any one of claims 57 to 63, wherein the cells from step (iii) or (vii) express CD4, CD8, and TCRαβ to the same extent as freshly harvested cells.

65. 58. The method of claim 57, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

66. The method of claim 645, wherein the PBMCs are added to the cell culture in step (iii) on any day from 9 to 17.

67. the effector in the larger TIL population of step (iii) or (vii).

67. The method of any one of claims 57 to 66, wherein the T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression compared to effector T cells and / or central memory T cells in said third cell population.

68. 68. The method of claim 67, wherein the effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.

69. The method of any one of claims 57 to 68, wherein the APC is an artificial APC (aAPC).

70. 70. The method of any one of claims 57 to 69, further comprising transducing the first TIL population with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

71. 71. The method of claim 70, wherein the transduction step occurs before step (i).

72. 72. The method of any one of claims 57-71, further comprising transducing the first TIL population with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

73. 73. The method of claim 72, wherein the transduction step occurs before step (i).

74. 76. The method of any one of claims 57 to 75, wherein the TILs are assayed for viability after step (vii).

75. 1. A method for assaying TILs, comprising: (i) obtaining a portion of the first cryopreserved TIL population; (ii) thawing said portion of said first cryopreserved TIL population; (iii) performing a first expansion culture by culturing the portion of the first TIL population in cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for a reREP period of at least 3 days to generate a second TIL population, wherein the portion from the first TIL population is compared to the second TIL population to determine a ratio of TIL numbers, and the ratio of TIL numbers in the second TIL population to TIL numbers in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to a patient based on the ratio of step (iii); and (v) when the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1 in step (iv), therapeutically administering the remainder of the first TIL population to the patient. A method comprising:

76. 76. The method of claim 75, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 50:

1.

77. 77. The method of claim 75 or 76, further comprising performing expansion of the entire first cryopreserved TIL population from step (i) by the method of any one of claims 1 to 76.

78. administering to said patient the entire first cryopreserved TIL population from step (i).

77. The method of claim 75 or 76, further comprising:

79. 78. The method of any one of claims 75-77, further comprising assessing the metabolic health of the second TIL population.

80. 79. The method of any one of claims 75-78, further comprising assessing the phenotype of the second TIL population.

81. 80. The method of any one of claims 75 to 79, wherein the antigen-presenting cells are allogeneic peripheral blood mononuclear cells.

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