Expansion culture of tumor-infiltrating lymphocytes from liquid tumors and their therapeutic use

Abstract

To provide the expansion culture of tumor infiltrating lymphocytes from liquid tumors and therapeutic uses thereof.SOLUTION: Methods of expansion-culturing tumor infiltrating lymphocytes (TILs), including peripheral blood lymphocytes and marrow infiltrating lymphocytes, from blood and / or bone marrow of patients with hematological malignancies, such as liquid tumors, including lymphomas and leukemias, and uses of such expansion-cultured TILs in the treatment of diseases such as cancers and hematological malignancies are disclosed herein.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 504,337, filed May 10, 2017; U.S. Provisional Patent Application No. 62 / 530,681, filed Jul. 10, 2017; U.S. Provisional Patent Application No. 62 / 550,398, filed Aug. 25, 2017; U.S. Provisional Patent Application No. 62 / 590,034, filed Nov. 22, 2017; U.S. Provisional Patent Application No. 62 / 621,462, filed Jan. 24, 2018; U.S. Provisional Patent Application No. 62 / 621,798, filed Jan. 25, 2018; and U.S. Provisional Patent Application No. 62 / 647,367, filed Mar. 23, 2018, all of which are hereby incorporated by reference in their entirety.

[0002] Field of the Invention

[0002] Disclosed herein are methods for expanding tumor - infiltrating lymphocytes (TIL) derived from the blood and / or bone marrow of patients having hematologic malignancies such as liquid tumors including lymphoma and leukemia, and compositions comprising populations of TIL obtained therefrom. Further disclosed herein is the therapeutic use of TIL expanded from the blood or bone marrow of patients having hematologic malignancies such as liquid tumors, including the treatment of such hematologic malignancies.

Background Art

[0003] Background of the Invention

[0003] The adoptive autologous transplantation of tumor-infiltrating lymphocytes (TILs) for the treatment of bulky and refractory cancers represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al, Nat.Rev. Immunol.2006, 6, 383-393. TILs are T cell-dominant, and IL-2-based TIL expansion culture followed by a "rapid expansion process" (REP) has become the preferred method for TIL expansion due to its speed and efficiency. Dudley, et al, Science 2002, 298, 850-54; Dudley, et al, J. Clin.Oncol.2005, 23, 2346-57; Dudley, et al, J. Clin.Oncol.2008, 26, 5233-39; Riddell, et al, Science 1992, 257, 238-41; Dudley, et al, J. Immunother.2003, 26, 332-42. Several approaches have been explored to improve the response to TIL therapy in melanoma and to expand TIL therapy to other types of tumors, but with limited success, and this area remains a challenge. Goff, et al., J. Clin.Oncol.2016, 34, 2389-97; Dudley, et al, J. Clin.Oncol.2008, 26, 5233-39; Rosenberg, et al, Clin.Cancer Res.2011, 17, 4550-57. Previous approaches to the expansion of TILs from B cell lymphoma have yielded insufficient results in which only two of twelve attempts at TIL proliferation provided potential activity against the tumor. Schwartzentruber, et al, Blood 1993, 82, 1204-1211. There is an urgent need to provide more effective treatment for many hematologic malignancies, including acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL).

Summary of the Invention

Means for Solving the Problems

[0004]

[0004] The present invention provides the surprising discovery that a TIL expansion culture process can result in an effective TIL population obtained from hematological malignancies such as liquid tumors including lymphoma or leukemia.

[0005] Summary of the Invention

[0005] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TIL), comprising: (a) optionally, pre-treating a patient with a regimen comprising at least one kinase inhibitor; (b) obtaining a tumor from the patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first population of TIL; (c) optionally, fragmenting or separating the tumor to obtain tumor fragments and contacting the tumor fragments with a first cell culture medium; (d) performing an initial expansion culture of the first population of TIL in the first cell culture medium to obtain a second population of TIL, wherein the second population of TIL is at least 5-fold more numerous than the first population of TIL, the first cell culture medium comprises IL-2, and the initial expansion culture is performed over a period of 21 days or less; (e) performing a second expansion culture of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the third population of TIL is at least 50-fold more numerous than the second population of TIL 7 days after the start of the second expansion culture, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the second expansion culture is performed over a period of 14 days or less; (f) recovering the third population of TIL; and (g) administering a therapeutically effective portion of the third population of TIL to a patient having cancer wherein the tumor is a liquid tumor and the cancer is a hematological malignancy.

[0006] In one embodiment, the present invention is a method of treating cancer using a population of tumor infiltrating lymphocytes (TILs), comprising: (a) optionally, pre-treating a patient with a regimen comprising at least one kinase inhibitor; (b) obtaining a tumor from the patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first population of TILs; (c) optionally, fragmenting or separating the tumor to obtain tumor fragments and contacting the tumor fragments with a first cell culture medium; (d) performing an initial expansion culture of the first population of TILs in the first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least five-fold greater in number than the first population of TILs, the first cell culture medium comprises IL-2, and the initial expansion culture is performed over a period of 21 days or less; (e) performing a second expansion culture of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs seven days after the start of the second expansion culture, the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the second expansion culture is performed over a period of 14 days or less; (f) recovering the third population of TILs; and (g) administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, the tumor is a liquid tumor, and the cancer is selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, relapsed and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myelogenous leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma, a method is provided.

[0007]

[0007] In one embodiment of the present invention, the method further comprises the addition of an ITK inhibitor. In one embodiment, the ITK inhibitor is added to the cell culture medium during at least one of steps (d) and (e). In one embodiment of the present invention, the ITK inhibitor is a covalent ITK inhibitor that binds covalently and irreversibly to ITK. In one embodiment of the present invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In another embodiment, the ITK inhibitor is selected from the group consisting of aminothiazole-based ITK inhibitors, benzimidazole-based ITK inhibitors, aminopyrimidine-based ITK inhibitors, 3-aminopyrid-2-one-based ITK inhibitors, indolylrhodanine-based ITK inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole inhibitors, and ITK inhibitors that target cysteine-442 in the ATP pocket. In another embodiment, the ITK inhibitor is ibrutinib, dasatinib, bosutinib, nilotinib, erlotinib BMS509744, CTA056, GSK2250665A, PF06465469 ((R)-3-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-(3-methyl-4-(1-methylethyl))benzamide), and combinations thereof. In another embodiment, the ITK inhibitor is ibrutinib. In another embodiment, the ITK inhibitor is (R)-3-(1-(1-acryloylpiperidin-3-yl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-(3-methyl-4-(1-methylethyl))benzamide. The aforementioned ITK inhibitors are commercially available from various suppliers including Tocris Bioscience, Inc. (Minneapolis, MN, USA), Selleckchem, Inc. (Houston, TX, USA), and AK Scientific, Inc. (Union City, CA, USA). In another embodiment, the ITK inhibitor is added at a concentration of about 0.1 nM to about 5 μM. In another embodiment, the ITK inhibitor is added at a concentration of about 0.1 nM to about 5 μM. In another embodiment, the ITK inhibitor is added at a concentration of about 0.1 nM to about 100 nM.In another embodiment, the ITK inhibitor is added at a concentration of about 0.5 nM to about 50 nM. In another embodiment, the ITK inhibitor is added at a concentration of about 1 nM to about 10 nM. In another embodiment, the ITK inhibitor is added at a concentration of about 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM.

[0008]

[0008] In one embodiment of the present invention, a method for expanding peripheral blood lymphocytes (PBL) from peripheral blood is a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from peripheral blood, wherein the sample is optionally cryopreserved; b. separating PBL from the sample by selecting and removing CD19+ B cells; c. optionally co-culturing the PBL with the CD19+ B cells; d. stimulating the PBL in the first cell culture medium in a gas-permeable container with IL-2 and anti-CD3 / anti-CD28 antibodies for a period of about 2 days to about 6 days; e. culturing the PBL from step (d) with IL-2 and anti-CD3 / anti-CD28 antibodies for a period of about 2 days to about 6 days; f. separating antibody-bound PBL from the culture from step (e); g. removing the antibody from the PBL separated in step (e); and h. recovering the PBL comprising.

[0009]

[0009] In one embodiment of the present invention, the method further comprises adding IL-2 after step (d) and replacing the first medium with a second cell culture medium. In another embodiment, the method further comprises adding IL-2 after step (e) and replacing the second medium with a third cell culture medium. In one embodiment, the first cell culture medium, the second cell culture medium, or the third cell culture medium is selected from the group consisting of CM-2, CM-4, and AIM-V. In another embodiment, the first and second cell culture media are the same. In another embodiment, the first and second cell culture media are different. In one embodiment of the present invention, one or more of the first, second, and third cell culture media are the same. In another embodiment, the first, second, and third cell culture media are all different.

[0010]

[0010] In one embodiment, the optional co-culture of the PBL and the CD19+ B cells is carried out over a period of 1 hour to 3 days.

[0011]

[0011] In one embodiment of the present invention, the ratio of T cells to B cells in step (c) is from about 0.1:1 to about 10:1 (B cells: T cells). In another embodiment, the ratio of B cells to T cells in step (c) is selected from the group consisting of 0.1:1, 1:1, and 10:1 (B cells: T cells).

[0012]

[0012] In one embodiment of the present invention, the starting cell number of PBL at the start of step (d) is at least about 1×10 5 ~ about 10×10 5 PBL. In another embodiment, the starting cell number of PBL at the start of step (d) is at least about 2.5×10 5 ~10×10 5 PBL. In another embodiment, the starting cell number of PBL at the start of step (d) is at least 5×10 5 PBL.

[0013]

[0013] In one embodiment of the present invention, the IL-2 in each of steps (c) and (d) is used at a concentration of about 1000 IU / mL to about 6000 IU / mL. In another embodiment, the IL-2 in each of steps (c) and (d) is used at a concentration of about 3000 IU / mL.

[0014]

[0014] In one embodiment of the present invention, the anti-CD3 / anti-CD28 antibody is coated on beads. In one embodiment of the present invention, the anti-CD3 / anti-CD28 antibody is DynaBeads®. In one embodiment, the method includes co-culturing anti-CD3 / anti-CD28 antibody beads with PBL at a bead:PBL ratio of about 1:1 in each of steps (c) and (d).

[0015]

[0015] In one embodiment of the present invention, the method includes adding an ITK inhibitor. In one embodiment, the ITK inhibitor is added in at least one of steps (c), (d), and (e). In one embodiment of the present invention, the ITK inhibitor is selected from the group consisting of aminothiazole-based ITK inhibitors, benzimidazole-based ITK inhibitors, aminopyrimidine-based ITK inhibitors, 3-aminopyrid-2-one-based ITK inhibitors, indolyl benzimidazole-based ITK inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole inhibitors, and ITK inhibitors targeting cysteine-442 in the ATP pocket. In another embodiment, the ITK inhibitor is ibrutinib, dasatinib, bosutinib, nilotinib, erlotinib BMS509744, CTA056, GSK2250665A, PF06465469, and combinations thereof. In another embodiment, the ITK inhibitor is ibrutinib.

[0016]

[0016] In an embodiment of the present invention, any of the aforementioned methods for preparing PBL is performed in a closed sterilization system.

[0017]

[0017] In one embodiment of the present invention, a method for expanding peripheral blood lymphocytes (PBL) from peripheral blood is a. Obtaining a sample of PBMCs from peripheral blood, wherein the sample is optionally cryopreserved; b. Separating PBLs from the sample by selecting and removing CD19+ B cells; c. Co-culturing the PBLs with the CD19+ B cells for a period of 4 days; d. Adding about 2.5×10 5 to about 5×10 5 cells to a gas-permeable container in CM-2 cell culture medium and stimulating the PBLs with 3000 IU / ml of IL-2 and anti-CD3 / anti-CD28 antibodies immobilized on beads for a period of about 4 days; e. Exchanging CM-2 with AIM-V cell culture medium and about 3000 IU / ml of additional IL-2; f. Culturing the PBLs from step (e) for an additional period of about 3 days with IL-2 and anti-CD3 / anti-CD28 antibodies immobilized on beads; g. Separating antibody-bound PBLs from the culture of step (f); h. Removing the antibody from the PBLs separated in step (g); and i. Recovering the PBLs comprising.

[0018]

[0018] In one embodiment of the present invention, a method for treating hematological malignancies is a. Obtaining a sample of PBMCs from the peripheral blood of a patient suffering from a hematological malignancy; b. Separating PBLs from the sample by selecting and removing CD19+ B cells; c. Optionally co-culturing the PBLs with the CD19+ B cells; d. Stimulating the PBLs in the first cell culture medium in a gas-permeable container with IL-2 and anti-CD3 / anti-CD28 antibodies for at least a period of about 4 days; e. Culturing the PBLs from step (d) for a period of 3 days with IL-2 and anti-CD3 / anti-CD28 antibodies; f. Separating antibody-bound PBLs from the culture of step (e); g. Removing the antibody from the PBLs separated in step (e); and h. Recovering the PBLs; and i. Administering the PBLs to a patient in a therapeutically effective amount to treat the blood malignancy comprising.

[0019]

[0019] In one embodiment of the present invention, the method further comprises obtaining a PBMC sample from a patient pre-treated with an ITK inhibitor. In one embodiment of the present invention, the ITK inhibitor is selected from the group consisting of aminothiazole-based ITK inhibitors, benzimidazole-based ITK inhibitors, aminopyrimidine-based ITK inhibitors, 3-aminopyrid-2-one-based ITK inhibitors, indolylrhodanine-based ITK inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole inhibitors, and ITK inhibitors targeting cysteine-442 within the ATP pocket. In one embodiment of the present invention, the ITK inhibitor is ibrutinib, BMS509744, CTA056, GSK2250665A, PF06465469, and combinations thereof. In another embodiment, the ITK inhibitor is ibrutinib. In another embodiment, the patient is pre-treated with at least three rounds of an ibrutinib regimen.

[0020]

[0020] In one embodiment of the present invention, the hematologic malignancy is selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, relapsed and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myelogenous leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma. In another embodiment, the hematologic malignancy is chronic lymphocytic leukemia (CLL). In one embodiment of the present invention, the PBL is about 0.1×10 9 ~ about 15×10 9 cells of PBL.

[0021]

[0021] In one embodiment of the present invention, the method for expanding myeloid infiltrating lymphocytes (MIL) from bone marrow is a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from bone marrow, wherein the sample is optionally cryopreserved; b. sorting CD3+, CD33+, CD20+ and CD14+ cell fractions (MIL fraction) and non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fractions (AML blast fraction); c. optionally disrupting the AML blast fraction; d. optionally adding the disrupted AML blast fraction to the MIL fraction at a cell number ratio of about 0.1:1 to about 10:1; e. culturing one or both cell fractions in a gas-permeable container in a first cell culture medium containing IL-2; f. stimulating the MIL with anti-CD3 / anti-CD28 antibodies to obtain expanded culture of the MIL; g. Restimulating the MIL with IL-2 and anti-CD3 / anti-CD28 antibodies over an additional period of about 2 to about 6 days; h. Culturing the MIL with additional IL-2 over an additional period of about 1 to about 3 days; and i. Recovering the MIL comprising.

[0022]

[0022] In one embodiment of the present invention, the method further comprises adding IL-2 after step (e) and replacing the medium with a second cell culture medium. In one embodiment, the first cell culture medium and the second cell culture medium are selected from the group consisting of CM-2, CM-4, and AIM-V. In another embodiment, the first and second cell culture media are the same. In another embodiment, the first and second cell culture media are different.

[0023]

[0023] In one embodiment, at the start of step (e), there are at least about 2×10 4 ~about 5×10 5 MIL in the gas-permeable container. In another embodiment, at the start of step (e), there are at least about 2.8×10 4 ~3.4×10 5 MIL in the gas-permeable container. In another embodiment, at the start of step (e), there are at least 5×10 5 MIL in the gas-permeable container.

[0024]

[0024] In one embodiment of the present invention, IL-2 is present at a concentration of 1000 IU / ml to 6000 IL / ml in step (e). In another embodiment, IL-2 is present at a concentration of about 6000 IU / ml. In another embodiment, IL-2 is present at a concentration of about 3000 IU / ml in step (g). In another embodiment, IL-2 is present at a concentration of about 3000 IU / ml in step (h).

[0025]

[0025] In one embodiment, the culturing in step (e) is carried out over a period of about 3 days. In one embodiment, the stimulation in step (f) is carried out over a period of about 4 days. In one embodiment, the stimulation in step (g) is carried out over a period of about 7 days.

[0026]

[0026] In one embodiment of the present invention, the optionally disrupted cell fraction is disrupted using a method selected from the group consisting of sonication, homogenization, vortexing, vibration, and lysis. In one embodiment of the present invention, the non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction (AML blast fraction) is lysed using a suitable lysis method including heat lysis, chemical lysis (such as organic alcohol), enzymatic lysis, and other cell lysis methods known in the art.

[0027]

[0027] In one embodiment of the present invention, the anti-CD3 / anti-CD28 antibody is coated on beads, and the MIL:bead ratio is about 1:1 in each of steps (f) and (g).

[0028]

[0028] In one embodiment of the present invention, the method is carried out in a closed sterilization system.

[0029]

[0029] In one embodiment of the present invention, a method for expanding bone marrow infiltrating lymphocytes (MIL) from bone marrow is a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from bone marrow, wherein the sample is optionally cryopreserved; obtaining; b. sorting CD3+, CD33+, CD20+ and CD14+ cell fractions (MIL cell fractions) and non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fractions (AML blast cell fractions); c. disrupting the AML blast cell fraction and adding the disrupted AML blast cell fraction to the MIL cell fraction at a cell number ratio of about 1:1; d. culturing the cell fractions in a gas-permeable container having a first cell culture medium containing about 6000 IU / ml of IL-2 over a period of about 3 days; e. adding anti-CD3 / anti-CD28 antibodies immobilized on beads to the cell culture at a ratio of approximately 1:1 (MIL: beads), and culturing the MIL and the antibodies for a period of approximately 1 day; f. replacing the first cell culture medium with a second cell culture medium containing an additional 3000 IU / ml of IL-2; g. culturing the antibodies and the MIL for an additional period of approximately 3 days; h. restimulating the MIL with IL-2 and anti-CD3 / anti-CD28 antibodies immobilized on beads for an additional period of at least approximately 4 days; i. replacing the second cell culture medium with a third cell culture medium containing an additional 3000 IU / ml of IL-2 for an additional period of at least approximately 3 days; j. recovering the MIL comprising.

[0030]

[0030] In one embodiment of the present invention, a method for treating hematological malignancies is a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from bone marrow, wherein the sample is optionally cryopreserved; b. sorting CD3+, CD33+, CD20+ and CD14+ cell fractions (MIL fraction) and non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fractions (AML blast fraction); c. optionally destroying the AML blast fraction; d. optionally adding the disrupted AML blast fraction to the MIL fraction at a cell number ratio of from about 0.1:1 to about 10:1; e. culturing one or both cell fractions in a gas-permeable container in a first cell culture medium containing IL-2; f. stimulating the sample with anti-CD3 / anti-CD28 antibodies; g. restimulating the MIL with IL-2 and anti-CD3 / anti-CD28 antibodies for an additional period of at least approximately 4 days; h. culturing the MIL with additional IL-2 for an additional period of at least approximately 3 days; i. recovering said MIL; and j. administering said MIL to a patient in a therapeutically effective amount for treating a hematological malignancy comprising.

[0031]

[0031] In one embodiment of the present invention, the hematological malignancy is acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, relapsed and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma. In another embodiment, the hematological malignancy is acute myeloid leukemia (AML). In one embodiment of the present invention, the MIL is administered in an amount of about 4×10 8 ~ about 2.5×10 9 MILs.

[0032] Brief Description of the Drawings

[0032] The foregoing summary and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0033]

Figure 1

[0033] Shows the pathological information of a lymphoma tumor.

Figure 2

[0034] Shows a comparison of different subsets of lymphoma and melanoma TILs, indicating that the effector memory (EM) subset of lymphoma TILs is significantly higher than the EM subset of melanoma TILs.

Figure 3

[0035] Comparison of different subsets of lymphoma and melanoma TILs is shown, indicating that the CD28+CD4+ subset of lymphoma TILs is significantly higher than these subsets of melanoma TILs.

Figure 4

[0036] Comparison of CD4+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs is shown, indicating differentiation markers. The red line in the graph represents the median. CM refers to central memory T cells, EM refers to effector memory T cells, and TEMRA refers to effector memory CD45RA+ T cells.

Figure 5

[0037] Comparison of CD8+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs is shown, indicating differentiation markers. The red line in the graph represents the median. CM refers to central memory T cells, EM refers to effector memory T cells, and TEMRA refers to effector memory CD45RA+ T cells.

Figure 6

[0038] Comparison of CD4+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs is shown, indicating exhaustion markers. The red line in the graph represents the median. LAG3 refers to lymphocyte activation gene 3, PD1 refers to programmed death 1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains.

Figure 7

[0039] Comparison of CD8+ T cell subsets of non-Hodgkin lymphoma TILs and melanoma TILs is shown, indicating exhaustion markers. The red line in the graph represents the median. LAG3 refers to lymphocyte activation gene 3, PD1 refers to programmed death 1, and TIGIT refers to T cell immunoreceptor with Ig and ITIM domains.

Figure 8

[0040] Comparison of cell types between non-Hodgkin lymphoma TILs and melanoma TILs is shown. NK refers to natural killer cells, and TCRab refers to cells expressing T cell receptors with alpha and beta chains.

Figure 9

[0041] Shows the results of the Bioluminescence Redirected Lysis Assay (BRLA).

Figure 10

[0042] Shows the results of the enzyme-linked immunosorbent assay (ELISA) for interferon-γ (IFN-γ) of lymphoma TILs versus melanoma TILs.

Figure 11

[0043] Shows the results of the enzyme-linked immunospot (ELIspot) assay for lymphoma TILs.

Figure 12

[0044] Shows the results of the ELIspot assay for melanoma TILs.

Figure 13

[0045] Shows the results of NANOSTRING NCOUNTER analysis, which indicates that lymphoma TILs express higher levels of RORC IL17A (TH17 phenotype) and GATA3 (Th2 phenotype) compared to melanoma TILs. Each gene is highlighted by the red box in the heat map.

Figure 14

[0046] Illustrates the TIL expansion culture and treatment process. Step 1 refers to the addition of 4 tumor fragments to 10 G-Rex 10 flasks. In Step 2, approximately 40×106 TILs or more are obtained. In Step 3, splitting occurs into 36 G-Rex 100 flasks for REP. In Step 4, the TILs are recovered by centrifugation. Fresh TIL product is obtained in Step 5 after a total process time of approximately 43 days, at which point the TILs can be injected into the patient.

Figure 15

[0047] Shows a treatment protocol for use with TILs obtained from the lymphoma of the present disclosure. Surgery (and tumor resection) is performed at the start, and lymphodepleting chemotherapy refers to myeloablative lymphodepletion with chemotherapy as described elsewhere in this specification.

Figure 16A

[0048] The results of flow cytometry analysis using the standard phenotype panel DF2 described in Example 4 below are shown. Tumor-infiltrating lymphocytes (TILs) from lymphoma and melanoma were stained using the standard phenotype panel DF2 as described in Example 4. The data shown represent different subpopulations of total CD4 and CD8 T cells in the TILs. The percentages of CD4 and CD8 cells in the naive T cell subset are shown. The P values were calculated using a two-sided Mann-Whitney test (unpaired). The mean percentages of the cell subsets are represented by horizontal bars.

Figure 16B

[0048] The results of flow cytometry analysis using the standard phenotype panel DF2 described in Example 4 below are shown. Tumor-infiltrating lymphocytes (TILs) from lymphoma and melanoma were stained using the standard phenotype panel DF2 as described in Example 4. The data shown represent different subpopulations of total CD4 and CD8 T cells in the TILs. The percentages of CD4 and CD8 cells in the central memory T cell subset (CM) are shown. The P values were calculated using a two-sided Mann-Whitney test (unpaired). The mean percentages of the cell subsets are represented by horizontal bars.

Figure 16C

[0048] The results of flow cytometry analysis using the standard phenotype panel DF2 described in Example 4 below are shown. Tumor-infiltrating lymphocytes (TILs) from lymphoma and melanoma were stained using the standard phenotype panel DF2 as described in Example 4. The data shown represent different subpopulations of total CD4 and CD8 T cells in the TILs. The percentages of CD4 and CD8 cells in the effector memory T cell subset (EM) are shown. The P values were calculated using a two-sided Mann-Whitney test (unpaired). The mean percentages of the cell subsets are represented by horizontal bars.

Figure 16D

[0048] The results of flow cytometry analysis using the standard phenotype panel DF2 described in Example 4 below are shown. TILs from lymphoma and melanoma were stained using the standard phenotype panel DF2 as described in Example 4. The data shown represent different subpopulations of total CD4 and CD8 T cells in TILs. The percentages of CD4 and CD8 cells in the terminally differentiated effector memory (TEMRA) T cell subset are shown. P values were calculated using a two-sided Mann-Whitney test (unpaired). The mean percentages of cell subsets are represented by horizontal bars.

Figure 17A

[0049] The results of flow cytometry analysis using the standard phenotype panel DF1 described in Example 4 below are shown. TILs from lymphoma and melanoma were stained using the standard phenotype panel DF1 as described in Example 4. The data shown represent different CD27+ subpopulations of total CD4 and CD9 T cells in TILs, which indicates a high proportion of co-stimulatory molecule CD28-expressing CD4 T cells in lymphoma TILs. P values were calculated using a two-sided Mann-Whitney test (unpaired).

Figure 17B

[0049] The results of flow cytometry analysis using the standard phenotype panel DF1 described in Example 4 below are shown. TILs from lymphoma and melanoma were stained using the standard phenotype panel DF1 as described in Example 4. The data shown represent different CD28+ subpopulations of total CD4 and CD9 T cells in TILs, which indicates a high proportion of co-stimulatory molecule CD28-expressing CD4 T cells in lymphoma TILs. P values were calculated using a two-sided Mann-Whitney test (unpaired).

Figure 18A

[0050] The results of an interferon gamma (IFN-γ) test performed according to Example 4 below are shown. The results using ELISpot are shown. ELISpot data are represented as IFN-γ-producing cells per 106 TILs.

Figure 18B

[0050] The results of an interferon gamma (IFN-γ) test conducted according to Example 4 below are shown. The results using ELISA are shown. ELISA data are expressed as IFN-γ levels in the supernatant of TIL cultures of 5×105 TIL / well measured on a logarithmic scale. P-values were calculated using a two-sided Mann-Whitney test (unpaired).

Figure 19A

[0051] Shows the lytic ability of TIL. Shows the LU50 of target cells normalized to 106 TIL at 4 hours in co-culture (TIL effector cells and GFP+P815 target cells).

Figure 19B

[0051] Shows the lytic ability of TIL. Shows the LU50 of target cells normalized to 106 TIL at 24 hours in co-culture (TIL effector cells and GFP+P815 target cells).

Figure 20A

[0052] Shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Shows the cytolytic activity of melanoma TILs against 526 allogeneic target cells. The data in Figure 20A are shown as the percentage of dead cells in a co-culture where the effector cell:target cell (E:T) ratio is 50:1.

Figure 20B

[0052] Shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Shows the cytolytic activity of lymphoma TILs against autologous tumor cells determined by 7-AAD uptake. The data in Figure 20B are shown as the percentage of dead cells in a co-culture where the effector cell:target cell (E:T) ratio is 50:1.

Figure 20C

[0052] Shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Represents the percentage of target cells killed induced by melanoma TILs.

Figure 20D

[0052] Shows the cytolytic activity of different TILs against allogeneic and autologous tumor types. Represents the percentage of target cells killed induced by lymphoma TILs at different E:T ratios.

Figure 21

[0053] A heatmap showing the gene expression profiles of lymphoma and melanoma TIL. The expression profiles were determined by the NanoString's 579-plex nCounter GX Human Immunology V2 CSO panel. The heatmap shows the fold change in the expression of a specific gene set of lymphoma TIL compared to melanoma TIL, suggesting higher expression of IL-17A and RORC from lymphoma-derived TIL. The cancers shown in this figure include follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma.

Figure 22

[0054] A schematic diagram showing the 2A process of the TIL preparation, recovery, and shipping schedule.

Figure 23

[0055] A flowchart showing the 2A process for preparing TIL.

Figure 24A - B

[0056] A flowchart demonstrating three different methods for expanding peripheral blood lymphocytes (PBL).

Figure 24C

[0056] A flowchart demonstrating three different methods for expanding peripheral blood lymphocytes (PBL).

Figure 25A - B

[0057] Represents three different methods for expanding marrow-infiltrating lymphocytes (MIL) from bone marrow.

Figure 25C

[0057] Represents three different methods for expanding marrow-infiltrating lymphocytes (MIL) from bone marrow.

Figure 26

[0058] A graph representing the expansion multiples of PBL isolated from fresh peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. The cryopreserved PBMC are from patients with CLL who were not treated (pre-Rx PBL) or treated (post-Rx PBL) with an ibrutinib regimen. Each point represents one patient. The red dots are patients in whom PBL was expanded using PBL method 1; the green dots are patients in whom PBL was expanded using PBL method 2; and the black dots are patients in whom PBL was expanded using PBL method 3.

Figure 27

[0059] Graph representing IFN-γ-producing cells of PBL isolated from fresh PBMC and cryopreserved PBMC. Pre-Rx PBL and post-Rx PBL within cryopreserved PBMC are also represented. Each point is a single patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 28

[0060] Graph showing the percentages of CD4+ and CD8+ T cell subsets in pre-Rx PBL and post-Rx PBL, using melanoma TIL as a comparator. Each point is a single patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 29A

[0061] Graph showing the comparison between CD4 memory subsets of pre-Rx PBL and post-Rx PBL, using melanoma TIL as a comparator. Data for naive (CCR7+ / CD45RA+) are shown. Each point is a single patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 29B

[0061] Graph showing the comparison between CD4 memory subsets of pre-Rx PBL and post-Rx PBL, using melanoma TIL as a comparator. Data for central memory t cells (CM) (CCR7+ / CD45RA-) are shown. Each point is a single patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 29C

[0061] Comparison of CD4 memory subsets of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator is shown. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 29D

[0061] Comparison of CD4 memory subsets of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator is shown. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 30A

[0061] Comparison of CD8 memory subsets of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator is shown. Data for naive (CCR7+ / CD45RA+) are shown. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 30B

[0061] Comparison of pre-Rx PBL and post-Rx PBL and CD8 memory subsets using melanoma TIL as a comparator is shown. Data for central memory T cells (CM) (CCR7+ / CD45RA-) are shown. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 30C

[0061] Comparison of the CD8 memory subsets of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator. Data on effector memory T cells (EM) (CCR7− / CD45RA−) are shown. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 30D

[0061] Comparison of the CD8 memory subsets of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator. Data on terminally differentiated effector memory cells (TEMRA) (CCR7− / CD45RA+) are shown. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 31A

[0062] Comparison of the CD27 subsets of the CD4 subsets of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 31B

[0062] Comparison of the CD27 subsets of the CD8 subsets of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 32A

[0063] Comparison of the CD28 subsets of the CD4 subset of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 32B

[0063] Comparison of the CD28 subsets of the CD8 subset of pre-Rx PBL and post-Rx PBL using melanoma TIL as a comparator. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 33A

[0064] Comparison of the LAG3+ subsets within the CD4 population of both pre-Rx PBL and post-Rx PBL. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 33B

[0064] Comparison of the LAG3+ subsets within the CD8 population of both pre-Rx PBL and post-Rx PBL. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 34A

[0065] Comparison of the PD1+ subsets within the CD4 population of both pre-Rx PBL and post-Rx PBL. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 34B

[0065] Shows the comparison of the PD1+ subset within the CD8 population for both pre-Rx PBL and post-Rx PBL. Each point represents one patient. Red dots are patients in whom PBL was expanded using PBL method 1; green dots are patients in whom PBL was expanded using PBL method 2; black dots are patients in whom PBL was expanded using PBL method 3.

Figure 35A

[0066] Shows the results of the cytolytic activity of pre-Rx PBL measured using the autologous tumor killing assay. Cytotoxicity is measured as LU50 (the number of PBL required to kill 50% of the target cells).

Figure 35B

[0066] Shows the results of the cytolytic activity of post-Rx PBL measured using the autologous tumor killing assay. Cytotoxicity is measured as LU50 (the number of PBL required to kill 50% of the target cells).

Figure 36A

[0067] Represents a graph of the magnification of MIL isolated from either the bone marrow (MIL) or peripheral blood (PBL) of AML patients. MIL1.1 was expanded using MIL method 1, MIL1.2 was expanded using MIL method 2, and MIL1.3 was expanded using MIL method 3. MIL2 and MIL3 were expanded using MIL method 3. All PBL were expanded using PBL method 3. The starting cell number of MIL1.3 was 138,000 cells, the starting cell number of MIL2 was 62,000, and the starting cell number of MIL3 was 28,000 cells. The starting cell number of PBL2 was 338,000, and the starting cell number of PBL3 was 336,000.

Figure 36B

Figure 37A

[0068] Shows IFN-γ-producing cells of MIL.

Figure 37B

[0068] Shows IFN-γ-producing cells of PBL.

Figure 38A

[0069] Represents a graph showing the T cell subsets of MIL isolated from AML patients. Shows the TCRαβ+ subset. PBL is shown on day 0 and day 14.

Figure 38B

[0069] Represents a graph showing the T cell subsets of MIL isolated from AML patients. Shows the CD4+ subset. PBL is shown on day 0 and day 14.

Figure 38C

[0069] Represents a graph showing the T cell subsets of MIL isolated from AML patients. Shows the CD8 subset. PBL is shown on day 0 and day 14.

Figure 38D

[0069] Represents a graph showing the T cell subsets of PBL isolated from AML patients. Shows the TCRαβ+ subset. PBL is shown on day 0 and day 14.

Figure 38E

[0069] Represents a graph showing the T cell subsets of PBL isolated from AML patients. Shows the CD4+ subset. PBL is shown on day 0 and day 14.

Figure 38F

Figure 39A

[0070] Graph representing the CD4 memory subset of MIL isolated from AML patients. Data for naive (CCR7+ / CD45RA+) are shown.

Figure 39B

[0070] Graph representing the CD4 memory subset of MIL isolated from AML patients. Data for central memory T cells (CM) (CCR7+ / CD45RA-) are shown.

Figure 39C

[0070] Graph representing the CD4 memory subset of MIL isolated from AML patients. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown.

Figure 39D

[0070] Graph representing the CD4 memory subset of MIL isolated from AML patients. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown.

Figure 40A

[0071] Graph representing the CD4 memory subset of PBL isolated from AML patients. Data for naive (CCR7+ / CD45RA+) are shown.

Figure 40B

[0071] Graph representing the CD4 memory subset of PBL isolated from AML patients. Data for central memory T cells (CM) (CCR7+ / CD45RA-) are shown.

Figure 40C

[0071] Graph representing the CD4 memory subset of PBL isolated from AML patients. Data for effector memory T cells (EM) (CCR7- / CD45RA-) are shown.

Figure 40D

[0071] Graph representing the CD4 memory subset of PBL isolated from AML patients. Data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown.

Figure 41A

[0072] Represents a graph showing the CD8 memory subset of MIL isolated from AML patients. Data of naive (CCR7+ / CD45RA+) are shown.

Figure 41B

[0072] Represents a graph showing the CD8 memory subset of MIL isolated from AML patients. Data of central memory T cells (CM) (CCR7+ / CD45RA-) are shown.

Figure 41C

[0072] Represents a graph showing the CD8 memory subset of MIL isolated from AML patients. Data of effector memory T cells (EM) (CCR7- / CD45RA-) are shown.

Figure 41D

[0072] Represents a graph showing the CD8 memory subset of MIL isolated from AML patients. Data of terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown.

Figure 42A

[0073] Represents a graph showing the CD8 memory subset of PBL isolated from AML patients. Data of naive (CCR7+ / CD45RA+) are shown.

Figure 42B

[0073] Represents a graph showing the CD8 memory subset of PBL isolated from AML patients. Data of central memory T cells (CM) (CCR7+ / CD45RA-) are shown.

Figure 42C

[0073] Represents a graph showing the CD8 memory subset of PBL isolated from AML patients. Data of effector memory T cells (EM) (CCR7- / CD45RA-) are shown.

Figure 42D

[0073] Represents a graph showing the CD8 memory subset of PBL isolated from AML patients. Data of terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+) are shown.

Figure 43A

[0074] Represents a graph showing the CD27 subsets of CD4 and CD8 cell populations of MIL.

Figure 43B

[0074] Represents a graph showing the CD27 subsets of CD4 and CD8 cell populations of PBL.

Figure 44A

[0075] Represents a graph showing the CD28 subset of the CD4 and CD8 cell populations of MIL.

Figure 44B

[0075] Represents a graph showing the CD28 subset of the CD4 and CD8 cell populations of PBL.

Figure 45A

[0076] Represents a graph showing the PD1+ subset of the CD4 and CD8 cell populations of MIL.

Figure 45B

[0076] Represents a graph showing the PD1+ subset of the CD4 and CD8 cell populations of PBL.

Figure 46A

[0077] Represents a graph showing the LAG3+ subset of the CD4 and CD8 cell populations of MIL.

Figure 46B

[0077] Represents a graph showing the LAG3+ subset of the CD4 and CD8 cell populations of PBL.

Figure 47

[0078] A timeline showing exemplary embodiments of PBL method 1 and PBL method 3. In this figure, the addition of IL-2 can occur at any point during the process, in an exemplary embodiment, on the region enclosed by parentheses.

Figure 48

[0079] A timeline showing an exemplary embodiment of MIL method 3. In this figure, the addition of IL-2 can occur at any point during the process, in an exemplary embodiment, on the region enclosed by parentheses.

Mode for Carrying Out the Invention

[0034] Brief Explanation of the Sequence Listing

[0080] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0081] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0082] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0083] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0084] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0085] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0086] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0087] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0036] DEFINITIONS

[0089] As used herein, the terms "co-administered", "co-administering", "administered in combination with", "administering in combination with", "simultaneous" and "concurrent" include the administration of two or more active pharmaceutical ingredients to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0037]

[0090] The term "in vivo" refers to events occurring within the body of a mammalian subject.

[0038]

[0091] The term "ex vivo" refers to events occurring outside the body of a mammalian subject in an artificial environment.

[0039]

[0092] The term "in vitro" refers to events occurring in a test system. In vitro assays include cell-based assays where viable or dead cells can be used, and may also include cell-free assays where intact cells are not used.

[0040]

[0093] The term "rapid expansion culture" means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold), more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold or 90-fold) or most preferably at least about 100-fold over a one-week period. Several rapid expansion culture protocols are described herein.

[0041]

[0094] The terms "fragmentation", "fragment" and "fragmented" as used herein to describe the process of disrupting a tumor include mechanical fragmentation methods such as disruption, slicing, division and mincing of tumor tissue and other methods of disrupting the physical structure of tumor tissue.

[0042]

[0095] The terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. Optionally, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. Antigen-presenting cells. The term "PBL" refers to peripheral blood lymphocytes and is T cells expanded from peripheral blood. The terms PBL and TIL are used interchangeably herein.

[0043]

[0096] The term "anti-CD3 antibody" refers to an antibody or a variant thereof, such as a monoclonal antibody, and includes human, humanized, chimeric or mouse antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3 and UCHT-1, also known as muromonab. Other anti-CD3 antibodies include, for example, otrexup, teprotumumab and visilizumab.

[0044]

[0097] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or a biosimilar or variant thereof that includes a human, humanized, chimeric, or murine antibody directed against the CD3 receptor in the T-cell antigen receptor of mature T cells, including OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and commercially available forms such as muromonab or its variants, conservative amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 have been deposited with the American Type Culture Collection and are assigned the ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 have also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and are assigned the catalog number 86022706.

[0045]

Table 1

[0046]

[0098] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, its conservative amino acid substitutions, glycoforms, biosimilars and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial) and recombinant IL-2 forms commercially supplied from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b) and other commercially available equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also includes pegylated forms of IL-2, including the pegylated IL2 prodrug NKTR-214 available from Nektar Therapeutics, South San Francisco, CA, USA, as described herein. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in US Patent Application Publication No. 2014 / 0328791A1 and International Publication No. 2012 / 065086A1 (the disclosures of which are incorporated herein by reference). Alternative forms of conjugated IL-2 suitable for use in the present invention are described in US Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference.Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0047]

Table 2

[0048]

[0099] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir.Res. 2001, 2, 66-70. When activated by IL-4, Th2 T cells subsequently produce further IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression and induces class switching to IgE and IgG1 expression from B cells. Recombinant human IL-15 suitable for use in the present invention is commercially available from a plurality of suppliers including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-4 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0049]

[0100] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin 7, which can be obtained from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the expression of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, in a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from a plurality of suppliers including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-7 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0050]

[0101] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15 and includes all forms of IL-15, including human and mammalian forms, its conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0051]

[0102] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, its conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat.Rev. Drug.Disc.2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 acts mainly on natural killer T cells and activated human CD4 +Produced by T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain with a molecular weight of 15.4 kDa and containing 132 amino acids. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, Catalog No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0052]

[0103] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. The use thereof in the therapeutic compositions of the present invention is contemplated, except when any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0053]

[0104] The term "antibody" in the singular and plural refers to immunoglobulins in their entirety and any antigen-binding fragments ("antigen-binding portions") or single chains thereof. "Antibody" further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V H and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as V L and a light chain constant region. The light chain constant region is composed of one domain, CL. The V H and V LThe region can be further subdivided into regions with hypervariability, which are referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs) and can be dispersed in more conserved regions (called framework regions (FRs)). Each V H and V L is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0054]

[0105] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can be further recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response that results in the activation of B lymphocytes and / or T lymphocytes. In some cases, this may require that the antigen contains or binds to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B epitopes and T epitopes). In some embodiments, an antigen preferably reacts with its corresponding antibody or TCR in a highly specific and selective manner typically and does not react with a number of other antibodies or TCRs induced by other antigens.

[0055]

[0106] The terms "monoclonal antibody", "mAb", "monoclonal antibody composition" or their plurals refer to preparations of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be made using knowledge and techniques in the art, which involve injecting an appropriate antigen into a test subject and then isolating hybridomas that express antibodies having the desired sequence or functional characteristics. DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. The DNA can be placed into an expression vector upon isolation and then transfected into host cells such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulin proteins by other methods to obtain the synthesis of monoclonal antibodies in recombinant host cells. The recombinant production of antibodies is described in more detail below.

[0056]

[0107] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the V L , V H , C L and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fd fragment consisting of the V H and CH1 domains; (iv) an Fv fragment consisting of the V L and V H domains of a single arm of the antibody; (v) a V H or VL Domain antibodies (dAb) fragments that can consist of a domain (Ward, et al, Nature, 1989, 341, 544-546), and (vi) isolated complementarity determining regions (CDRs) are included. Further, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but they can be linked by a synthetic linker that allows them to be made as a single protein chain that forms a monovalent molecule known as a single-chain Fv (scFv), the V L and V H region pair; see, for example, Bird, et al., Science 1988, 242, 423-426; and Huston, et al, Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be included within the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0057]

[0108] As used herein, the term "human antibody" is intended to include antibodies having a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Further, when the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted into human framework sequences.

[0058]

[0109] The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has a variable region in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In one embodiment, a human monoclonal antibody is obtained from a transgenic non-human animal, such as a transgenic mouse, and is produced by a hybridoma containing B cells having a genome that includes a human heavy chain transgene and a light chain transgene fused to immortalized cells.

[0059]

[0110] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (such as mice) of the human immunoglobulin genes or hybridomas prepared therefrom (further described below), (b) antibodies isolated from host cells transformed to express human antibodies, such as transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework region and the CDR region are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if transgenic animals are used for human Ig sequences), and thus the amino acid sequences of the V H and V L regions are derived from and related to the V H and V L sequences of the human germline, while being sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0060]

[0111] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region gene (e.g., IgM or IgG1).

[0061]

[0112] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably in this specification with the term "antibody that specifically binds to an antigen".

[0062]

[0113] The term "human antibody derivative" refers to any variant form of a human antibody that includes an antibody and another active pharmaceutical ingredient or a conjugate with an antibody. The terms "conjugate", "antibody-drug conjugate", "ADC" or "immunoconjugate" refer to an antibody or a fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

[0063]

[0114] The terms "humanized antibody", "humanized antibodies", and "humanized" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted into a human framework sequence. Further modifications of framework regions within the human framework sequence may be made. A humanized form of a non-human (e.g., mouse) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable regions of the recipient have been replaced by residues from the 15 hypervariable regions (donor antibody) of a non-human species, such as a mouse, rat, rabbit, or non-human primate, that have the desired specificity, affinity, and potency. In some examples, the Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody includes substantially all of at least one, typically two, variable domains, where all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequence. A humanized antibody optionally also includes at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, et al, Nature 1986, 321, 522-525; Riechmann, et al, Nature 1988, 332, 323-329; and Presta, Curr.Op.Struct.Biol 1992, 2, 593-596. The antibodies described herein may be modified to use any Fc variant known to confer improvement (e.g., reduction) in effector function and / or FcR binding.Fc variants can include any one of the amino acid substitutions disclosed in, for example, WO 1988 / 07089 A1, WO 1996 / 14339 A1, WO 1998 / 05787 A1, WO 1998 / 23289 A1, WO 1999 / 51642 A1, WO 99 / 58572 A1, WO 2000 / 09560 A2, WO 2000 / 32767 A1, WO 2000 / 42072 A2, WO 2002 / 44215 A2, WO 2002 / 060919 A2, WO 2003 / 074569 A2, WO 2004 / 016750 A2, WO 2004 / 029207 A2, WO 2004 / 035752 A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 A1, WO 2006 / 047350 A2, and WO 2006 / 085967 A2; and U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784, the disclosures of which are incorporated herein by reference.

[0064]

[0115] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0065]

[0116] "Diabody" is a small antibody fragment having two antigen-binding sites. The fragment is a polypeptide chain in which the same (V H -V L or V L -V H) within the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on the other chain, generating two antigen-binding sites. Diabodies are described more fully, for example, in European Patent No. 404,097, International Publication No. 93 / 11161; and Bolliger, et al, Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0066]

[0117] The term "glycosylation" refers to modified derivatives of antibodies. Aglycosylated antibodies lack glycosylation. Glycosylation can be modified, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby removing glycosylation at that site. As described in U.S. Patent Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies with modified glycosylation types can be produced, such as hypofucosylated antibodies having a reduced amount of fucosyl residues or antibodies having an increased bisecting GlcNac structure. Such modified glycosylation patterns have been demonstrated to increase the ability of the antibody. Such carbohydrate modifications can be achieved, for example, by altering the glycosylation machinery to express the antibody in a host cell. Cells with modified glycosylation machinery have been described in the art and can be used as host cells for expressing the recombinant antibodies of the present invention, thereby producing antibodies with modified glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha(1,6)fucosyltransferase), and thus the antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were generated by targeting the disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (e.g., U.S. Patent Application Publication No. 2004 / 0110704 or Yamane-Ohnuki, et al, Biotechnol. Bioeng., 2004, 87, 614-622).As another example, European Patent No. 1,176,195 describes a cell line having a functionally disrupted FUT8 gene encoding a fucosyltransferase such that the antibody expressed in such a cell line exhibits hypofucosylation by reducing or eliminating an alpha1,6-linkage-related enzyme, and also a cell line having low or no enzyme activity of adding fucose to N-acetylglucosamine that binds to the Fc region of the antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). International Publication No. 03 / 035835 describes a mutant CHO cell line, Lec13 cell, which has a reduced ability to bind fucose to the carbohydrate linked to Asn(297) and results in hypofucosylation of the antibody expressed in its host cell (see also Shields, et al, J. Biol Chem.2002, 277, 26733-26740). International Publication No. 99 / 54342 describes a cell line (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) engineered to express a glycoprotein-modifying glycosyltransferase such that the antibody expressed in the engineered cell line exhibits an increased bisecting GlcNac structure that results in an increase in the ADCC activity of the antibody (see also Umana, et al., Nat.Biotech.1999, 77, 176-180). Alternatively, the fucose residues of the antibody can be cleaved using a fucosidase enzyme. For example, alpha-L-fucosidase, a fucosidase, removes fucosyl residues derived from antibodies as described in Tarentino, et al., Biochem.1975, 14, 5516-5523.

[0067]

[0118] "Pegylation" refers to a modified antibody or a fragment thereof that reacts with PEG, such as a reactive ester or aldehyde derivative of polyethylene glycol (PEG), under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Pegylation can, for example, increase the biological (e.g., serum) half-life of an antibody. Preferably, pegylation is effected via an acylation or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C 10 )alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated can be an aglycosylated antibody. Methods of pegylation are known in the art and can be applied to the antibodies of the present invention, as described, for example, in European Patent Nos. 0154316 and 0401384 and U.S. Patent No. 5,824,778, the disclosures of each of which are incorporated herein by reference.

[0068]

[0119] The term "fusion protein" or "fusion polypeptide" refers to a protein that combines the properties of two or more individual proteins. Such a protein has at least two heterologous polypeptides covalently linked either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically linked from the C-terminus to the N-terminus, but can also be linked from C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order and can include any two or more or both of the constituent polypeptides. This term includes conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs, and immunogenic fragments of the antigens that make up the fusion protein. The fusion proteins of the present disclosure can also include additional copies of the component antigens or immunogenic fragments thereof. The fusion protein can include one or more binding domains that bind to each other and further bind to an Fc domain such as an IgG Fc domain. The fusion proteins can be further linked together to mimic monoclonal antibodies and provide six or more binding domains. The fusion proteins can be produced by recombinant methods as known in the art. The preparation of fusion proteins is known in the art and is described, for example, in International Publication Nos. WO 1995 / 027735 A1, WO 2005 / 103077 A1, WO 2008 / 025516 A1, WO 2009 / 007120 A1, WO 2010 / 003766 A1, WO 2010 / 010051 A1, WO 2010 / 078966 A1, US Patent Application Publication Nos. 2015 / 0125419 A1 and 2016 / 0272695 A1, and US Patent No. 8,921,519, the disclosures of each of which are incorporated herein by reference.

[0069]

[0120] The term "heterologous" when used with respect to a nucleic acid or protein moiety indicates that the nucleic acid or protein contains two or more sub-sequences that are not found in nature in the same relationship to each other. For example, a nucleic acid is typically recombinantly produced and has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another source or coding regions from different sources. Similarly, a heterologous protein indicates that the protein contains two or more sub-sequences that are not found in nature in the same relationship to each other (e.g., a fusion protein).

[0070]

[0121] The term "conservative amino acid substitution" means an amino acid sequence modification that does not abrogate the binding of an antibody or fusion protein to its antigen. Conservative amino acid substitutions include the substitution of one amino acid of one class with another amino acid of the same class, where the classes are defined by common physicochemical amino acid side chain properties and high substitution frequencies in naturally occurring homologous proteins, as determined, for example, by a standard Dayhoff frequency exchange matrix or a BLOSUM matrix. Six general classes of amino acid side chains are classified, including class I (Cys); class II (Ser, Thr, Pro, Ala, Gly); class III (Asn, Asp, Gln, Glu); class IV (His, Arg, Lys); class V (Ile, Leu, Val, Met); and class VI (Phe, Tyr, Trp). For example, the substitution of Asp with another class III residue, such as Asn, Gln or Glu, is a conservative substitution. Thus, predicted non-essential amino acid residues in an antibody are preferably substituted with another amino acid residue from the same class. Methods for identifying amino acid conservative substitutions that do not eliminate antigen binding are well known in the art (see, for example, Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al, Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).

[0071]

[0122] In connection with two or more nucleic acids or polypeptides, the terms "sequence identity", "percent identity" and "sequence percent identity" (or synonyms thereof, e.g., "99% identical") refer to two or more sequences or subsequences that, when compared for maximal correspondence and aligned (introducing gaps if necessary) without considering conservative amino acid substitutions as part of sequence identity, are the same or have a specified percentage of the same nucleotide or amino acid residues. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art for obtaining an alignment of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the National Center for Biotechnology Information of the United States government. The comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign available from DNASTAR are additional publicly available software programs that can be used to align sequences. One of ordinary skill in the art can determine appropriate parameters for maximal alignment with a particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0072]

[0123] As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein having an amino acid sequence different from that of a reference antibody due to one or more substitutions, deletions and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of the reference antibody. Conservative substitutions can include, for example, substitutions of amino acids that are similarly charged or uncharged. A variant retains the ability to specifically bind to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

[0073]

[0124] A nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly shown sequence. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues. Batzer, et al, Nucleic Acid Res.1991, 79, 5081;Ohtsuka, et al, J. Biol.Chem.1985, 260, 2605-2608;Rossolini, et al., Mol.Cell.Probes 1994, 8, 91-98. The term "nucleic acid" is used interchangeably with cDNA, mRNA, oligonucleotide and polynucleotide.

[0074]

[0125] The term "biosimilar" means a biological product, including a monoclonal antibody or a protein, which is highly similar to an approved reference biological product in the United States, notwithstanding minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of the product's safety, purity, and potency. Further, a similar biological or "biosimilar" medicine is a biological medicine similar to another biological medicine that has already been approved for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by regulatory authorities in other countries and regions. A biological preparation or biological medicine is a medicine made or derived from a biological source such as bacteria or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is Aldesleukin (PROLEUKIN), the protein approved by the pharmaceutical regulatory authority for Aldesleukin is a "biosimilar to" Aldesleukin or "its biosimilar" of Aldesleukin. In Europe, a similar biological or "biosimilar" medicine is a biological medicine similar to another biological medicine that has already been approved for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological uses in Europe is Article 6 of the amended Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. Thus, in Europe, a biosimilar can be approved under Article 6 of the Regulation (EC) No 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC, and the approval can be granted or be the subject of an approval application. In Europe, the original biological medicine that has already been approved is sometimes called the "reference medicine". Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guidelines for similar biological medicines. Further, product-specific guidelines, including guidelines for monoclonal antibody biosimilars, are provided by the EMA for each product and are publicly available on its website.The biosimilars described in this specification may be similar to the reference medicinal product in terms of quality characteristics, biological activity, mechanism of action, safety profile and / or efficacy. Furthermore, the biosimilar may be used or intended to be used for treating the same condition as the reference medicinal product. Accordingly, the biosimilars described in this specification may be considered to have quality characteristics similar or very similar to those of the reference medicinal product. Alternatively or additionally, the biosimilars described in this specification may be considered to have biological activity similar or very similar to that of the reference medicinal product. Alternatively or additionally, the biosimilars described in this specification may be considered to have a safety profile similar or very similar to that of the reference medicinal product. Alternatively or additionally, the biosimilars described in this specification may be considered to have efficacy similar or very similar to that of the reference medicinal product. As described herein, biosimilars in Europe are compared to the reference medicinal product authorized by the EMA. However, in some cases, the biosimilar may be compared to a biological medicinal product authorized outside the European Economic Area (non-EEA authorized "comparator") in a particular study. Such tests include, for example, certain clinical trials and in vivo non-clinical trials. As used herein, the term "biosimilar" also relates to biological medicinal products that have been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (e.g., antigen-binding portions) and fusion proteins. The protein biosimilar may have an amino acid sequence with minor modifications to the amino acid structure (including, for example, amino acid deletions, additions and / or substitutions) that do not significantly affect the function of the polypeptide. The biosimilar may include an amino acid sequence having at least 97% sequence identity, such as 97%, 98%, 99% or 100%, to the amino acid sequence of its reference medicinal product. The biosimilar may have one or more post-translational modifications different from those of the reference medicinal product, including, but not limited to, glycosylation, oxidation, deamidation and / or cleavage, provided that the differences do not result in a change in the safety and / or efficacy of the medicinal product. The biosimilar may have the same or a different glycosylation pattern as the reference medicinal product.Although not exclusive, biosimilars may have different glycosylation patterns, particularly when the differences are intended to address or address concerns regarding the safety of the reference medicinal product. Furthermore, biosimilars may deviate from the reference medicinal product in terms of, for example, its strength, pharmaceutical form, formulation, excipients and / or presentation, provided that the safety and efficacy of the medicinal product are not compromised. Biosimilars may include, for example, differences in the pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles when compared to the reference medicinal product, but are still considered to be sufficiently similar to the reference medicinal product to be approved or considered suitable for approval. In certain circumstances, biosimilars exhibit different binding properties compared to the reference medicinal product, where the different binding properties are not considered by regulatory authorities such as the EMA to be a barrier to approval as a similar biological product. The term "biosimilar" is also used synonymously by regulatory agencies in other countries and regions.

[0075]

[0126] The term "hematological malignancy" refers to cancers and tumors of the hematopoietic and lymphatic tissues of mammals, including but not limited to blood, bone marrow, lymph nodes and lymphatic tissues. Hematological malignancies can lead to the formation of "liquid tumors". Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin lymphoma and non-Hodgkin lymphoma. The term "B-cell hematological malignancy" refers to hematological malignancies that affect B cells.

[0076]

[0127] The term "liquid tumor" refers to an abnormal mass of cells that is inherently fluid. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other blood malignancies. TILs obtained from liquid tumors, including those present in the bone marrow, may also be referred to herein as marrow-infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including those circulating in peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only by the tissue type from which the cells are derived.

[0077]

[0128] The term "biopsy" refers to any medical procedure used to obtain cancer cells, including bone marrow biopsies.

[0078]

[0129] The term "acute myeloid leukemia" or "AML" refers to cancer of the myeloid blood cell line, which is also known in the art as acute myelogenous leukemia and acute non-lymphocytic leukemia. AML is a liquid tumor, although some manifestations of AML, including extramedullary findings such as chloromas, exhibit characteristics of solid tumors and are classified herein as liquid tumors.

[0079]

[0130] As used herein, the term "microenvironment" can refer to the solid or blood tumor microenvironment as a whole or to individual subsets of cells within the microenvironment. The tumor microenvironment as used herein refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from the host immune system, foster treatment resistance, and provide a niche for successful metastatic seeding," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that should be recognized by T cells, but elimination of tumors by the immune system is rare due to immune suppression by the microenvironment.

[0080]

[0131] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein that is sufficient to achieve the intended use, including but not limited to treating a disease. The therapeutically effective amount can vary depending on the intended use (in vitro or in vivo), the subject being treated and the disease state (e.g., the weight, age and gender of the subject), the severity of the disease state or the method of administration. This term also applies to the dosage that induces a specific response (e.g., a decrease in platelet adhesion and / or cell migration) in target cells. The specific dosage will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered and the physical delivery system by which the compound is carried.

[0081]

[0132] "Therapeutic effect", as the term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit. A prophylactic effect includes delaying or precluding the occurrence of a disease or condition, delaying or precluding the onset of symptoms of a disease or condition, delaying, arresting or reversing the progression of a disease or condition or any combination thereof.

[0082]

[0133] The terms "treatment", "treating", "treat" and the like refer to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic in the sense of completely or partially preventing a disease or its symptoms, and / or therapeutic in the sense of partially or completely curing a disease and / or an adverse effect caused by the disease. "Treatment", as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) alleviating the disease, i.e., causing regression of the disease and / or alleviating one or more symptoms of the disease. "Treatment" is also intended to encompass the delivery of an agent for providing a pharmacological effect even in the absence of a disease or disorder. For example, "treatment" includes the delivery of a composition that can induce an immune response or confer immunity in the absence of a disease state, such as in the case of a vaccine.

[0083]

[0134] The terms "QD", "qd" or "q.d." mean once a day, once daily or every day. The terms "BID", "bid" or "b.i.d." mean twice a day, twice daily or every two days. The terms "TID", "tid" or "t.i.d." mean three times a day, three times daily or every three days. The terms "QID", "qid" or "q.i.d." mean four times a day, four times daily or every four days.

[0084]

[0135] As used herein, "tumor-infiltrating lymphocytes" or "TILs" refers to a population of cells that are originally obtained as white blood cells and have migrated from the bloodstream of a subject to infiltrate a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from patient tissue samples as outlined herein (and may be referred to as "freshly harvested") and "secondary TILs" include, but are not limited to, any population of TIL cells that have been expanded or propagated as discussed herein, including bulk TILs, expanded culture TILs ("REP TILs"), and "reREP TILs" as discussed herein.

[0085]

[0136] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and affect treatment. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be defined functionally by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs can be further characterized by potency - for example, a TIL can be considered potent if, for example, interferon (IFN) release is higher than about 50 pg / mL, higher than about 100 pg / mL, higher than about 150 pg / mL, or higher than about 200 pg / mL.

[0086]

[0137] As used herein, "cryopreserved TIL" (or cryopreserved MIL or PBL) means that TIL of any of primary, bulk or expanded culture (REP TIL) is treated and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere in this specification, including in the Examples. For clarity, "cryopreserved TIL" is distinguishable from cryopreserved tissue samples that can be used as a source of primary TIL.

[0087]

[0138] As used herein, "thawed cryopreserved TIL" (or thawed MIL or PBL) means a population of TIL that was previously cryopreserved and then treated to return to a temperature above room temperature, including, but not limited to, cell culture temperature or a temperature at which TIL can be administered to a patient.

[0088]

[0139] As used herein, "population of cells" (including TIL) means several cells that share a common trait. Generally, the population is generally in the range of 1×10 6 ~ 1×10 10 and different TIL populations include different numbers. For example, the initial expansion of primary TIL in the presence of IL-2 results in a bulk TIL population of approximately 1×10 8 cells. REP expansion is generally performed to provide a population of 1.5×10 9 ~ 1.5×10 10 cells for injection.

[0089]

[0140] Generally, TIL is first obtained from a patient tumor sample ("primary TIL"), then expanded to a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally phenotyped and metabolic parameters are determined as indicators of TIL health.

[0090]

[0141] Generally, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0091]

[0142] Generally, as discussed herein, TILs are prepared by first obtaining a primary TIL population from a tumor excised from a patient, as discussed herein (the "primary cell population" or "first cell population"). This is followed by an initial bulk expansion culture utilizing culturing the cells with IL-2, and a second cell population is formed (which may also be referred to herein as the "bulk TIL population" or "second population").

[0092]

[0143] The term "cytotoxic lymphocyte" includes cytotoxic T (CTL) cells (CD8 + cytotoxic T lymphocytes and CD4 + T-helper lymphocytes), natural killer T (NKT) cells, and natural killer (NK) cells. Cytotoxic lymphocytes can include, for example, peripheral blood-derived αβTCR-positive T cells or γδTCR-positive T cells activated by tumor-associated antigens and / or transduced with a tumor-specific chimeric antigen receptor or T cell receptor, as well as tumor-infiltrating lymphocytes (TILs).

[0093]

[0144] The term "central memory T cell" refers to a subset of T cells that are CD45RO+ in humans and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells mainly secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment of the blood and are proportionally concentrated in lymph nodes and tonsils in humans.

[0094]

[0145] The term "effector memory T cell" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+ but lack constitutive expression of CCR7 (CCR7lo) and have heterogeneous or low expression of CD62L (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. The transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, after antigen stimulation. Effector memory T cells are predominant in the CD8 compartment of the blood and are proportionally concentrated in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large 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 with the methods of the present invention. Examples of closed systems include, but are not limited to, sealed G containers. When tumor segments are added to a closed system, the system is not opened to the external environment until the TILs are ready to be administered to the patient.

[0095]

[0146] In some embodiments, the method of the present disclosure grows tumor tissue or cells from tumor tissue in a standard laboratory medium (including, without limitation, RPMI) and treats it with reagents such as irradiated feeder cells and anti-CD3 antibodies to increase the number of TILs and / or enrich a population of cells including desired cell surface markers or other structural, biochemical, or functional characteristics, etc., to achieve a desired effect, further including a "pre-REP" stage. The pre-REP stage can utilize experimental-grade reagents (under the assumption that the experimental-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, during the pre-REP stage, the disclosed TLR agonists and / or peptides or peptidomimetics can be included in the culture medium. The pre-REP culture can include IL-2 in some embodiments. The present invention, in preferred embodiments, surprisingly leads to the expansion of memory T cell subsets, including memory effector T cell subsets, and / or a significant enhancement of glycolytic respiration when compared to thawed cryopreserved TILs for freshly harvested TILs or restimulated TILs (sometimes referred to herein as "reTILs"), and relates to a novel method of enhancing REP with one or more additional restimulation protocols, also referred to herein as the "restimulation rapid expansion culture protocol" or "reREP". That is, by using the reREP procedure on cryopreserved TILs, patients can receive highly metabolically active and healthy TILs, which can lead to better outcomes.

[0096]

[0147] When an "anti-tumor effective amount", "tumor-suppressing effective amount", or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician considering individual differences in age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). Generally, a pharmaceutical composition containing the genetically modified cytotoxic lymphocytes described herein is 10 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 、10 5 ~10 10 、105 ~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 said that it can be administered at a dosage of (including all integer values within these ranges) cells / kg body weight. The genetically modified cytotoxic lymphocyte composition can also be administered multiple times at these dosages. The genetically modified cytotoxic lymphocytes can be administered by using infusion techniques generally known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0097]

[0148] For the avoidance of doubt, it is intended that in this specification, any particular feature (e.g., integer, property, value, use, disease, formula, compound or group) described in connection with a particular aspect, embodiment or example of the invention is to be understood as applicable to any other aspect, embodiment or example described herein, unless incompatible. Accordingly, such features can be used, if desired, in combination with any of the definitions, claims or embodiments defined herein. All features (including the appended claims, abstract and drawings) disclosed herein and / or all steps of any method or process so disclosed can be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. The invention is not limited to any details of any of the disclosed embodiments. The invention extends to any novel or novel combination of the features (including the appended claims, abstract and drawings) disclosed herein or to any novel or novel combination of the steps of any method or process so disclosed.

[0098]

[0149] The terms "about" and "approximately" mean within a statistically significant range of values. Such ranges can be within one order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variations encompassed by the term "about" or "approximately" depend on the particular system under study and can be readily understood by those skilled in the art. Further, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics need not be exact and need not be exact, but rather reflect tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art, and can be approximate and / or larger or smaller as appropriate. In general, dimensions, sizes, formulations, parameters, shapes, or other quantities or features are "about" or "approximately", whether or not explicitly stated to be so. Note that embodiments of very different sizes, shapes, and dimensions can employ the described mechanisms.

[0099]

[0150] In the original and amended forms, when used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the subject matter of the claims with respect to what is excluded from the scope of the claims if there are additional elements or steps of the claims not recited. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional unrecited elements, method, steps, or materials. The term "consisting of" excludes any element, step, or material other than those expressly recited in the claims and, in the case of materials, ordinary impurities associated with the expressly recited materials. The term "consisting essentially of" limits the claims to the specified elements, steps, or materials and those that do not substantially affect the basic and novel characteristics of the invention recited in the claims. All compositions, methods, and kits described herein embodying the present invention can be more specifically defined in alternative embodiments by any of the transitional terms "comprising," "consisting essentially of," and "consisting of."

[0100] Embodiments of a method for expanding and culturing therapeutic T cells comprising peripheral blood (PBL) and / or bone marrow (MIL) A method for expanding and culturing peripheral blood lymphocytes (PBL) from peripheral blood

[0151] PBL Method 1. In one embodiment of the present invention, PBL is expanded in culture using the processes described herein. In one embodiment of the present invention, the method includes obtaining a PBMC sample from whole blood. In one embodiment, the method includes enriching T cells by separating pure T cells from PBMCs using negative selection of the non-CD19+ fraction. On day 0, the pure T cells are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 1:1 (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is stimulated again with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, the PBLs are harvested, the beads are removed, the PBLs are counted, and phenotyped. In one embodiment, the method includes enriching T cells by separating pure T cells from PBMCs using magnetic bead-based negative selection of the non-CD19+ fraction.

[0101]

[0152] In one embodiment of the present invention, PBL method 1 is implemented as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. T cells are separated using a Human PanT cell isolation kit and an LS column (Miltenyi Biotec). The separated T cells are counted and seeded at 5×10 5 cells per well in a GRex 24-well plate and co-cultured with DynaBeads® (anti-CD3 / anti-CD28) at a ratio of 1:1 with 3000 IU / ml of IL-2 in a total of 8 ml of CM2 medium per well. On day 4, the medium in each well is exchanged from CM2 to AIM-V containing 3000 IU / ml of fresh IL-2. On day 7, the expanded cells are harvested, counted, and then cultured in a GRex 10M flask at 15×10 6Cultivate with individual cells. On the 11th day, replace the medium with CM-4 medium supplemented with 3000 IU / ml of fresh IL-2. On the 14th day, use a DynaMag Magnet (DynaMag™-15) to remove the DynaBeads® and count the cells.

[0102]

[0153] In one embodiment of the present invention, PBL method 1 is carried out as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMC is counted. T cells are separated using a Human PanT cell isolation kit and an LS column (Miltenyi Biotec). The separated T cells are counted and seeded at 5×10 5 cells per well in a total of 8 ml of CM2 medium and co-cultured with DynaBeads® (anti-CD3 / anti-CD28) at a ratio of 3000 IU of IL-2 to 1:1. On the 4th day, replace the medium in each well from CM2 to AIM-V containing 3000 IU / ml of fresh IL-2. On the 7th day, collect the PBL, count it, and then re-seed it at 1×106 cells per well in a total of 8 ml of AIM-V medium with 3000 IU / ml of IL-2 and DynaBeads® at a ratio of 1:1 (beads:cells). On the 11th day, replace the medium with CM-4 medium supplemented with 3000 IU / ml of fresh IL-2. On the 14th day, use a DynaMag Magnet (DynaMag™-15) to remove the DynaBeads® and count the cells.

[0103]

[0154] PBL Method 2. In one embodiment of the present invention, PBL is expanded using PBL Method 2, which includes obtaining a PBMC sample from whole blood. T cells from PBMC are enriched by incubating PBMC at 37°C for at least 3 hours and then separating the non-adherent cells. The non-adherent cells are expanded in culture in the same manner as PBL Method 1, i.e., on day 0, the non-adherent cells are cultured with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a ratio of 1:1 (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is stimulated again with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, PBL is harvested, the beads are removed, PBL is counted, and phenotyped.

[0104]

[0155] In one embodiment of the present invention, PBL Method 2 is carried out as follows: On day 0, the cryopreserved PMBC sample is thawed, and PBMC cells are seeded at 6 million cells per well in a 6-well plate in CM-2 medium and incubated at 37°C for 3 hours. After 3 hours, the non-adherent cells, which are PBL, are removed and counted. PBL is 6 cultured in each well of a GRex 24-well plate with 1×10 6Cultivate with individual cells. On the 11th day, replace the medium with CM-4 medium and supplement with fresh IL-2 (3000 IU / ml). On the 14th day, use DynaMag™ Magnet (DynaMag™-15) to remove DynaBeads® and count the cells.

[0105]

[0156] In one embodiment of the present invention, PBL method 2 is implemented as follows: On day 0, thaw the cryopreserved PMBC sample, seed the PBMC cells in a 6-well plate in CM-2 medium at 6 million cells per well, and incubate at 37°C for 3 hours. After 3 hours, remove and count the non-adherent cells, which are PBL. PBL is 1×10 6 cells, and culture in each well of a GRex 24-well plate with anti-CD3 / anti-CD28 DynaBeads® at a bead:cell ratio of 1:1 and 3000 IU / ml of IL-2 in a total of 7 ml of CM-2 medium. On the 4th day, replace the medium in each well with AIM-V medium and 3000 IU / ml of fresh IL-2. On the 7th day, collect and count the expanded cells, and then culture in a total of 8 ml of AIM-V medium with 3000 IU / ml of IL-2 and DynaBeads® at a ratio of 1:1 (T cell:bead) at 1×10 6 cells per well of a GRex 24-well plate. On the 11th day, replace the medium with CM-4 medium and supplement with fresh IL-2 (3000 IU / ml). On the 14th day, use DynaMag™ Magnet (DynaMag™-15) to remove DynaBeads® and count the cells.

[0106]

[0157] PBL method 3. In one embodiment of the present invention, PBL is expanded using PBL method 3 which includes obtaining a PBMC sample from peripheral blood. B cells are separated using CD19+ selection, and T cells are selected using negative selection of the non-CD19+ fraction of the PBMC sample. On day 0, T cells and B cells are co-cultured with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a ratio of 1:1 (beads: cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is stimulated again with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a ratio of 1:1 (beads: cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, PBL is recovered, the beads are removed, PBL is counted, and phenotyped.

[0107]

[0158] In one embodiment of the present invention, PBL method 3 is carried out as follows: On day 0, cryopreserved PBMCs derived from peripheral blood are thawed and counted. CD19+ B cells are sorted using CD19 Multisort Kit, Human (Miltenyi Biotec). Among the non-CD19+ cell fraction, T cells are purified using Human PanT cell isolation kit and LS column (Miltenyi Biotec). T cells (PBL) and B cells are co-cultured at different ratios in a Grex 24-well plate in about 8 ml of CM2 medium in the presence of about 3000 IU / ml of IL-2. The ratio of B cells: T cells is 0.1:1, 1:1, and 10:1. The T cell / B cell co-culture is stimulated with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a ratio of 1:1 (beads: cells). On day 4, the medium is changed from CM2 to AIM-V medium, and additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the cells are recovered, counted, and transferred to a new Grex 24-well plate with AIM-V medium, at about 1.5×10 5 ~ about 4×10 5Reseed in the cell range of individual cells and stimulate with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 1:1 (beads: cells) together with an additional 3000 IU / mL of IL-2. On day 14, the DynaBeads® are removed using a DynaMag™ Magnet (DynaMag™-15) and the cells are counted.

[0108]

[0159] In one embodiment, PBMCs are isolated from a whole blood sample. In one embodiment, the PBMC sample is used as a starting material for expanding PBLs. In one embodiment, the sample is cryopreserved prior to the expansion culture process. In another embodiment, a fresh sample is used as a starting material for expanding PBLs. In one embodiment of the present invention, T cells are isolated from PBMCs using methods known in the art. In one embodiment, T cells are isolated using a Human Pan T cell isolation kit and an LS column. In one embodiment of the present invention, T cells are isolated from PBMCs using an antibody selection method known in the art, such as CD19 negative selection.

[0109]

[0160] In an embodiment of the present invention, the process is carried out over about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days or about 14 days. In another embodiment, the process is carried out over about 7 days. In another embodiment, the process is carried out over about 14 days.

[0110]

[0161] In one embodiment of the present invention, PBMC is cultured with anti-CD3 / anti-CD28 antibody. In one embodiment, any available anti-CD3 / anti-CD28 product is useful in the present invention. In one embodiment of the present invention, the commercially available product used is DynaBeads®. In one embodiment, DynaBeads® is cultured with PBMC at a ratio of 1:1 (beads:cells). In another embodiment, the antibody is DynaBeads® cultured with PBMC at a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1 (beads:cells). In one embodiment of the present invention, the antibody culture step and / or the step of restimulating cells with the antibody is carried out over a period of about 2 to about 6 days, about 3 to about 5 days or about 4 days. In one embodiment of the present invention, the antibody culture step is carried out over a period of about 2 days, 3 days, 4 days, 5 days or 6 days.

[0111]

[0162] In one embodiment, the PBMC sample is cultured with IL-2. In one embodiment of the present invention, the cell culture medium used for the expansion culture of PBL from PBMC contains IL-2 at a concentration selected from the group consisting of about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,000 IU / mL, about 1,100 IU / mL, about 1,200 IU / mL, about 1,300 IU / mL, about 1,400 IU / mL, about 1,500 IU / mL, about 1,600 IU / mL, about 1,700 IU / mL, about 1,800 IU / mL, about 1,900 IU / mL, about 2,000 IU / mL, about 2,100 IU / mL, about 2,200 IU / mL, about 2,300 IU / mL, about 2,400 IU / mL, about 2,500 IU / mL, about 2,600 IU / mL, about 2,700 IU / mL, about 2,800 IU / mL, about 2,900 IU / mL, about 3,000 IU / mL, about 3,100 IU / mL, about 3,200 IU / mL, about 3,300 IU / mL, about 3,400 IU / mL, about 3,500 IU / mL, about 3,600 IU / mL, about 3,700 IU / mL, about 3,800 IU / mL, about 3,900 IU / mL, about 4,000 IU / mL, about 4,100 IU / mL, about 4,200 IU / mL, about 4,300 IU / mL, about 4,400 IU / mL, about 4,500 IU / mL, about 4,600 IU / mL, about 4,700 IU / mL, about 4,800 IU / mL, about 4,900 IU / mL, about 5,000 IU / mL, about 5,100 IU / mL, about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL and about 10,000 IU / mL.

[0112]

[0163] In one embodiment of the present invention, the starting cell number of PBMCs for the expansion culture process is from about 25,000 to about 1,000,000, from about 30,000 to about 900,000, from about 35,000 to about 850,000, from about 40,000 to about 800,000, from about 45,000 to about 800,000, from about 50,000 to about 750,000, from about 55,000 to about 700,000, from about 60,000 to about 650,000, from about 65,000 to about 600,000, from about 70,000 to about 550,000, preferably from about 75,000 to about 500,000, from about 80,000 to about 450,000, from about 85,000 to about 400,000, from about 90,000 to about 350,000, from about 95,000 to about 300,000, from about 100,000 to about 250,000, from about 105,000 to about 200,000 or from about 110,000 to about 150,000. In one embodiment of the present invention, the starting cell number of PBMCs is about 138,000, 140,000, 145,000 or a number exceeding that. In another embodiment, the starting cell number of PBMCs is about 28,000. In another embodiment, the starting cell number of PBMCs is about 62,000. In another embodiment, the starting cell number of PBMCs is about 338,000. In another embodiment, the starting cell number of PBMCs is about 336,000.

[0113]

[0164] In one embodiment of the present invention, the cells are grown in a GRex 24-well plate. In one embodiment of the present invention, an equivalent well plate is used. In one embodiment, the starting material for the expansion culture is about 5×10 5 T cells per well. In one embodiment of the present invention, there are 1×10 6 cells per well. In an embodiment of the present invention, the number of cells per well is a number sufficient to seed the well and expand the T cells in culture.

[0114]

[0165] In one embodiment of the present invention, the magnification of PBL is from about 20% to about 100%, 25% to about 95%, 30% to about 90%, 35% to about 85%, 40% to about 80%, 45% to about 75%, 50% to about 100% or 25% to about 75%. In one embodiment of the present invention, the magnification is about 25%. In another embodiment of the present invention, the magnification is about 50%. In another embodiment, the magnification is about 75%.

[0115]

[0166] In one embodiment of the present invention, additional IL-2 can be added to the culture for 1 day or more throughout the process. In one embodiment of the present invention, additional IL-2 is added on the 4th day. In one embodiment of the present invention, additional IL-2 is added on the 7th day. In one embodiment of the present invention, additional IL-2 is added on the 11th day. In other embodiments, additional IL-2 is added on the 4th, 7th and / or 11th days. In one embodiment of the present invention, the cell culture medium can be replaced within 1 day or more throughout the cell culture process. In one embodiment, the cell culture medium is replaced on the 4th, 7th and / or 11th days of the process. In one embodiment of the present invention, PBL is cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days together with additional IL-2. In an embodiment of the present invention, PBL is cultured for a period of 3 days after each addition of IL-2.

[0116]

[0167] In one embodiment, the cell culture medium is replaced at least once during the method. In one embodiment, the cell culture medium is replaced simultaneously with the addition of additional IL-2. In another embodiment, the cell culture medium is replaced on at least 1 day out of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th or 14th day. In one embodiment of the present invention, the cell culture medium used throughout the method can be the same or different. In one embodiment of the present invention, the cell culture medium is CM-2, CM-4 or AIM-V.

[0117]

[0168] In one embodiment of the present invention, the T cells can be restimulated with anti-CD3 / anti-CD28 antibodies for one or more days through a 14-day expansion culture process. In one embodiment, the T cells are restimulated on day 7. In one embodiment, a GRex 10M flask is used for the restimulation step. In one embodiment of the present invention, an equivalent flask is used.

[0118]

[0169] In one embodiment of the present invention, DynaBeads® are removed using a DynaMag™ Magnet, the cells are counted, and the cells are analyzed using the phenotypic and functional assays further described in the following examples. In one embodiment of the present invention, the antibodies are isolated from PBL or MIL using methods known in the art. In any of the foregoing embodiments, magnetic bead-based selection of TIL, PBL, or MIL is used.

[0119]

[0170] In one embodiment of the present invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify non-adherent cells. In one embodiment of the present invention, the incubation time is about 3 hours. In one embodiment of the present invention, the temperature is about 37 degrees Celsius. The non-adherent cells are then expanded using the above process.

[0120]

[0171] In one embodiment of the present invention, PBMCs are obtained from patients treated with ibrutinib or another ITK or kinase inhibitor such as the ITK and kinase inhibitors described elsewhere herein. In one embodiment of the present invention, the ITK inhibitor is a covalent ITK inhibitor that binds covalently and irreversibly to ITK. In one embodiment of the present invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK. In one embodiment of the present invention, PBMCs are obtained from patients treated with ibrutinib or another ITK inhibitor, including the ITK inhibitors described elsewhere herein, prior to obtaining a PBMC sample for use in any of the aforementioned methods, including PBL method 1, PBL method 2, or PBL method 3. In one embodiment of the present invention, the ITK inhibitor treatment has been administered at least once, at least twice, at least three times, or more than that. In one embodiment of the present invention, PBLs expanded from patients pretreated with ibrutinib or another ITK inhibitor contain fewer LAG3+ and PD-1+ cells than cells expanded from patients not pretreated with ibrutinib or another ITK inhibitor. In one embodiment of the present invention, PBLs expanded from patients pretreated with ibrutinib or another ITK inhibitor contain increased levels of IFNγ production compared to cells expanded from patients not pretreated with ibrutinib or another ITK inhibitor. In one embodiment of the present invention, PBLs expanded from patients pretreated with ibrutinib or another ITK inhibitor contain increased lytic activity at a lower effector:target cell ratio compared to cells expanded from patients not pretreated with ibrutinib or another ITK inhibitor. In one embodiment of the present invention, patients pretreated with ibrutinib or another ITK inhibitor have a higher fold expansion compared to untreated patients.

[0121]

[0172] In one embodiment of the present invention, the method includes the step of adding an ITK inhibitor to a cell culture. In one embodiment, the ITK inhibitor is added on one or more of day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 of the process. In one embodiment, the ITK inhibitor is added on a day during the method in which the cell culture medium is changed. In one embodiment, the ITK inhibitor is added on day 0 and when the cell culture medium is changed. In one embodiment, the ITK inhibitor is added during the method in which IL-2 is added. In one embodiment, the ITK inhibitor is added on day 0, day 4, day 7, and optionally day 11 of the method. In one embodiment of the present invention, the ITK inhibitor is added on day 0 and day 7 of the method. In one embodiment of the present invention, the ITK inhibitor is known in the art. In one embodiment of the present invention, the ITK inhibitor is as described in other parts of this specification.

[0122]

[0173] In one embodiment of the present invention, the ITK inhibitor is used in the method at a concentration of about 0.1 nM to about 5 μM. In one embodiment, the ITK inhibitor is used in the method at a concentration of about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM.

[0123]

[0174] In one embodiment of the present invention, the method includes the step of adding an ITK inhibitor when the PBMC is derived from a patient who has no prior exposure to treatment with an ITK inhibitor such as ibrutinib.

[0124]

[0175] In some embodiments, the PBMC sample is from a subject or patient optionally pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, 1 year or more. In another embodiment, the PBMC is from a patient currently receiving an ITK inhibitor regimen such as ibrutinib.

[0125]

[0176] In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor and resistant to treatment with a kinase inhibitor or an ITK inhibitor such as ibrutinib.

[0126]

[0177] In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but no longer receiving treatment with a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but no longer receiving treatment with a kinase inhibitor or an ITK inhibitor and not treated for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year or more. In another embodiment, the PBMC is from a patient with prior exposure to an ITK inhibitor but not treated for at least 3 months, at least 6 months, at least 9 months or at least 1 year.

[0127]

[0178] In an embodiment of the present invention, on day 0, cells are selected for CD19+ and appropriately sorted. In one embodiment of the present invention, the selection is performed using antibody-conjugated beads. In one embodiment of the present invention, pure T cells are separated from PBMC on day 0. In one embodiment of the present invention, on day 0, CD19+ B cells and pure T cells are co-cultured with anti-CD3 / anti-CD28 antibodies for at least 4 days. In one embodiment of the present invention, on day 4, IL-2 is added to the culture. In one embodiment of the present invention, on day 7, the culture is restimulated with anti-CD3 / anti-CD28 antibodies and additional IL-2. In one embodiment of the present invention, PBL is collected on day 14.

[0128]

[0179] In one embodiment of the present invention, for patients not pretreated with ibrutinib or other ITK inhibitors, 10 - 15 ml of buffy coat yields approximately 5×10 9 individual PBMCs, which is approximately 5.5×10 7 starting cell materials and approximately 1.1×10 9 individual PBLs at the end of the expansion culture process. In one embodiment of the present invention, approximately 54×10 6 individual PBMCs yield approximately 6×10 5 starting materials and approximately 1.2×10 8 individual MILs (an approximately 205-fold expansion).

[0129]

[0180] In one embodiment of the present invention, for patients pretreated with ibrutinib or other ITK inhibitors, the expansion culture process yields approximately 20×10 9 individual PBLs. In one embodiment of the present invention, 40.3×10 6 individual PBMCs yield approximately 4.7×10 5 starting cell materials and approximately 1.6×10 8 individual PBLs (an approximately 338-fold expansion).

[0130]

[0181] In one embodiment of the present invention, the clinical dose of PBL useful in the present invention for patients with chronic lymphocytic leukemia (CLL) is approximately 0.1×10 9 to approximately 15×109 individual PBLs, about 0.1×10 9 ~ about 15×10 9 individual PBLs, about 0.12×10 9 ~ about 12×10 9 individual PBLs, about 0.15×10 9 ~ about 11×10 9 individual PBLs, about 0.2×10 9 ~ about 10×10 9 individual PBLs, about 0.3×10 9 ~ about 9×10 9 individual PBLs, about 0.4×10 9 ~ about 8×10 9 individual PBLs, about 0.5×10 9 ~ about 7×10 9 individual PBLs, about 0.6×10 9 ~ about 6×10 9 individual PBLs, about 0.7×10 9 ~ about 5×10 9 individual PBLs, about 0.8×10 9 ~ about 4×10 9 individual PBLs, about 0.9×10 9 ~ about 3×10 9 individual PBLs or about 1×10 9 ~ about 2×10 9 individual PBLs.

[0131]

[0182] In any of the foregoing embodiments, PBMCs can be obtained from a whole blood sample by apheresis, from a buffy coat, or from any other method known in the art for obtaining PBMCs.

[0132] Method for expanding and culturing bone marrow infiltrating lymphocytes (MIL) from bone marrow-derived PBMCs

[0183] MIL method 1. In one embodiment of the present invention, a method for expanding and culturing MIL from bone marrow-derived PBMC is described. In one embodiment of the present invention, the method is carried out over 14 days. In one embodiment, the method includes obtaining bone marrow PBMC and cryopreserving the PBMC. On day 0, the PBMC is cultured with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a ratio of 1:1 (beads: cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is stimulated again with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a 1:1 ratio (beads: cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 14, the MIL is recovered, the beads are removed, and the MIL is optionally counted and phenotyped.

[0133]

[0184] In one embodiment of the present invention, MIL method 1 is carried out as follows: On day 0, a cryopreserved PBMC sample derived from bone marrow is thawed and the PBMC is counted. The PBMC is co-cultured in a GRex 24-well plate with anti-CD3 / anti-CD28 antibody (DynaBeads®) at a ratio of 1:1 at about 8 ml of CM-2 cell culture medium (composed of RPMI-1640, human AB serum, 1-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium) per well in the presence of 3000 IU / ml of IL-2 at 5×10 5 cells per well. On day 4, the cell culture medium is replaced with AIM-V supplemented with 3000 IU / ml of additional IL-2. On day 7, the expanded MIL is counted. 1×10 6Transfer the cells into a new Grex 24-well plate and culture them with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 1:1 in approximately 8 ml of AIM-V medium per well in the presence of 3000 IU / ml of IL-2. On day 11, exchange the cell culture medium from AIM-V to CM-4 (composed of AIM-V medium, 2 mM Glutamax, and 3000 IU / ml of IL2). On day 14, remove the DynaBeads® using a DynaMag Magnet (DynaMag™ 15) and count the MIL.

[0134]

[0185] MIL method 2. In one embodiment of the present invention, the method is carried out over 7 days. In one embodiment, the method includes obtaining PMBCs derived from bone marrow and cryopreserving the PBMCs. On day 0, culture the PBMCs with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 3:1 (beads:cells) and 3000 IU / ml of IL-2. On day 7, collect the MIL, remove the beads, optionally count the MIL, and phenotype it.

[0135]

[0186] In one embodiment of the present invention, the MIL method 2 is carried out as follows: On day 0, the cryopreserved PBMC sample is thawed and the PBMCs are counted. The PBMCs are co-cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 1:1 with 5×10 5 cells per well in approximately 8 ml of CM-2 cell culture medium (composed of RPMI-1640, human AB serum, 1-glutamine, 2-mercaptoethanol, gentamicin sulfate, AIM-V medium) per well in the presence of 3000 IU / ml of IL-2 in a Grex 24-well plate. On day 7, remove the DynaBeads® using a DynaMag Magnet (DynaMag™ 15) and count the MIL.

[0136]

[0187] MIL method 3. In one embodiment of the present invention, the method includes obtaining PBMCs from bone marrow. On day 0, PBMCs are selected, sorted for CD3+ / CD33+ / CD20+ / CD14+, the non-CD3+ / CD33+ / CD20+ / CD14+ cell fraction is sonicated, and a portion of the sonicated cell fraction is returned to and added to the selected cell fraction. IL-2 is added to the cell culture at 3000 IU / ml. On day 3, the PBMCs are cultured with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 1:1 (beads:cells) and 3000 IU / ml of IL-2. On day 4, additional IL-2 is added to the culture at 3000 IU / ml. On day 7, the culture is stimulated again with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a 1:1 ratio (beads:cells), and 3000 IU / ml of additional IL-2 is added to the culture. On day 11, IL-2 is added to the culture at 3000 IU / ml. On day 14, the MIL is recovered, the beads are removed, and the MIL is optionally counted and phenotyped.

[0137]

[0188] In one embodiment of the present invention, the MIL method 3 is carried out as follows: On day 0, a sample of cryopreserved PBMCs is thawed and the PBMCs are counted. The cells are stained with CD3, CD33, CD20 and CD14 antibodies and sorted using S3e cell sorting (Bio-Rad). The cells are sorted into two fractions: an immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) and an AML blast cell fraction (non-CD3+CD33+CD20+CD14+). The number of cells from the AML blast cell fraction approximately equal to the number of cells from the immune cell fraction (or MIL fraction) seeded in a Grex 24-well plate is suspended in 100 ul of medium and sonicated. In this example, from about 2.8×10 4 ~ about 3.38×10 5Take individual cells, suspend them in 100 ul of CM2 medium, and sonicate for 30 seconds. Add 100 ul of the sonicated AML blast cell fraction to the immune cell fraction in a Grex24 well plate. The immune cells are present in approximately 8 ml of CM-2 cell culture medium per well in the presence of 6000 IU / ml of IL-2 at a quantity of approximately 2.8×10 4 ~ approximately 3.38×10 5 cells per well and are cultured for approximately 3 days together with a portion of the AML blast cell fraction. On day 3, add anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 1:1 to each well and culture for approximately 1 day. On day 4, replace the cell culture medium with AIM-V supplemented with 3000 IU / ml of additional IL-2. On day 7, count the expanded MIL. Approximately 1.5×10 5 ~ 4×10 5 cells per well are transferred to a new Grex 24 well plate and cultured in approximately 8 ml of AIM-V medium per well in the presence of 3000 IU / ml of IL-2 together with anti-CD3 / anti-CD28 antibodies (DynaBeads®) at a ratio of 1:1. On day 11, replace the cell culture medium from AIM-V to CM-4 (supplemented with 3000 IU / ml of IL-2). On day 14, remove the DynaBeads® using a DynaMag Magnet (DynaMag™ 15), and optionally count the MIL.

[0138]

[0189] In one embodiment of the present invention, PBMC is obtained from bone marrow. In one embodiment, PBMC is obtained from bone marrow through apheresis, aspiration, needle biopsy, or other similar means known in the art. In one embodiment, PBMC is fresh. In another embodiment, PBMC is cryopreserved.

[0139]

[0190] In one embodiment of the present invention, the method is carried out over approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, or approximately 14 days. In another embodiment, the method is carried out over approximately 7 days. In another embodiment, the method is carried out over approximately 14 days.

[0140]

[0191] In one embodiment of the present invention, PBMCs are cultured with anti-CD3 / anti-CD28 antibodies. In one embodiment, any available anti-CD3 / anti-CD28 product is useful in the present invention. In one embodiment of the present invention, the commercially available product used is DynaBeads®. In one embodiment, DynaBeads® are cultured with PBMCs at a ratio of 1:1 (beads:cells). In another embodiment, the antibody is DynaBeads® cultured with PBMCs at a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1 (beads:cells). In any of the foregoing embodiments, magnetic bead-based selection of the immune cell fraction (or MIL fraction) (CD3+CD33+CD20+CD14+) or the AML blast cell fraction (non-CD3+CD33+CD20+CD14+) is used. In one embodiment of the present invention, the antibody culture step and / or the step of restimulating the cells with the antibody is carried out over a period of about 2 to about 6 days, about 3 to about 5 days or about 4 days. In one embodiment of the present invention, the antibody culture step is carried out over a period of about 2 days, 3 days, 4 days, 5 days or 6 days.

[0141]

[0192] In one embodiment of the present invention, the ratio of the number of cells from the AML blast cell fraction to the number of cells from the immune cell fraction (or MIL fraction) is from about 0.1:1 to about 10:1. In another embodiment, the ratio is from about 0.1:1 to about 5:1, from about 0.1:1 to about 2:1 or about 1:1. In one embodiment of the present invention, the AML blast cell fraction is optionally disrupted to break cell aggregation. In one embodiment, the AML blast cell fraction is disrupted using sonication, homogenization, cell lysis, vortexing or vibration. In another embodiment, the AML blast cell fraction is disrupted using sonication. In one embodiment of the present invention, the non-CD3+, non-CD33+, non-CD20+, non-CD14+ cell fraction (AML blast fraction) is lysed using a suitable lysis method including heat lysis, chemical lysis (such as organic alcohol), enzymatic lysis and other cell lysis methods known in the art.

[0142]

[0193] In one embodiment of the present invention, the cells from the AML blast cell fraction are suspended at a concentration of about 0.2×10 5 to about 2×10 5 cells per 100 μL and added to the cell culture together with the immune cell fraction. In another embodiment, the concentration is about 0.5×10 5 to about 2×10 5 cells per 100 μL, about 0.7×10 5 to about 2×10 5 cells per 100 μL, about 1×10 5 to about 2×10 5 cells per 100 μL or about 1.5×10 5 to about 2×10 5 cells per 100 μL.

[0143]

[0194] In one embodiment, the PBMC sample is cultured with IL-2. In one embodiment of the present invention, the cell culture medium used for the expansion culture of MIL contains IL-2 at a concentration selected from the group consisting of about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,000 IU / mL, about 1,100 IU / mL, about 1,200 IU / mL, about 1,300 IU / mL, about 1,400 IU / mL, about 1,500 IU / mL, about 1,600 IU / mL, about 1,700 IU / mL, about 1,800 IU / mL, about 1,900 IU / mL, about 2,000 IU / mL, about 2,100 IU / mL, about 2,200 IU / mL, about 2,300 IU / mL, about 2,400 IU / mL, about 2,500 IU / mL, about 2,600 IU / mL, about 2,700 IU / mL, about 2,800 IU / mL, about 2,900 IU / mL, about 3,000 IU / mL, about 3,100 IU / mL, about 3,200 IU / mL, about 3,300 IU / mL, about 3,400 IU / mL, about 3,500 IU / mL, about 3,600 IU / mL, about 3,700 IU / mL, about 3,800 IU / mL, about 3,900 IU / mL, about 4,000 IU / mL, about 4,100 IU / mL, about 4,200 IU / mL, about 4,300 IU / mL, about 4,400 IU / mL, about 4,500 IU / mL, about 4,600 IU / mL, about 4,700 IU / mL, about 4,800 IU / mL, about 4,900 IU / mL, about 5,000 IU / mL, about 5,100 IU / mL, about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL, and about 10,000 IU / mL.

[0144]

[0195] In one embodiment of the present invention, additional IL-2 can be added to the culture for one day or more throughout the method. In one embodiment of the present invention, the additional IL-2 is added on the 4th day. In one embodiment of the present invention, the additional IL-2 is added on the 7th day. In one embodiment of the present invention, the additional IL-2 is added on the 11th day. In another embodiment, the additional IL-2 is added on the 4th day, 7th day and / or 11th day. In one embodiment of the present invention, the MIL is cultured for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days together with the additional IL-2. In one embodiment of the present invention, the MIL is cultured for a period of 3 days after each addition of IL-2.

[0145]

[0196] In one embodiment, the cell culture medium is exchanged at least once during the method. In one embodiment, the cell culture medium is exchanged simultaneously with the addition of additional IL-2. In another embodiment, the cell culture medium is exchanged on at least one day of day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13 or day 14. In one embodiment of the present invention, the cell culture medium used throughout the method may be the same or different. In one embodiment of the present invention, the cell culture medium is CM-2, CM-4 or AIM-V. In one embodiment of the present invention, the step of exchanging the cell culture medium on day 11 is optional. In one embodiment of the present invention, the starting cell number of PBMCs for the expansion culture process is about 25,000 to about 1,000,000, about 30,000 to about 900,000, about 35,000 to about 850,000, about 40,000 to about 800,000, about 45,000 to about 800,000, about 50,000 to about 750,000, about 55,000 to about 700,000, about 60,000 to about 650,000, about 65,000 to about 600,000, about 70,000 to about 550,000, preferably about 75,000 to about 500,000, about 80,000 to about 450,000, about 85,000 to about 400,000, about 90,000 to about 350,000, about 95,000 to about 300,000, about 100,000 to about 250,000, about 105,000 to about 200,000 or about 110,000 to about 150,000. In one embodiment of the present invention, the starting cell number of PBMCs is about 138,000, 140,000, 145,000 or more. In another embodiment, the starting cell number of PBMCs is about 28,000. In another embodiment, the starting cell number of PBMCs is about 62,000. In another embodiment, the starting cell number of PBMCs is about 338,000. In another embodiment, the starting cell number of PBMCs is about 336,000.

[0146]

[0197] In one embodiment of the present invention, the magnification of MIL is about 20% to about 100%, 25% to about 95%, 30% to about 90%, 35% to about 85%, 40% to about 80%, 45% to about 75%, 50% to about 100% or 25% to about 75%. In one embodiment of the present invention, the magnification is about 25%. In another embodiment of the present invention, the magnification is about 50%. In another embodiment, the magnification is about 75%.

[0147]

[0198] In one embodiment of the present invention, MIL is expanded and cultured from 10 to 50 ml of bone marrow aspirate. In one embodiment of the present invention, 10 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 20 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 30 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 40 ml of bone marrow aspirate is obtained from a patient. In another embodiment, 50 ml of bone marrow aspirate is obtained from a patient.

[0148]

[0199] In one embodiment of the present invention, the number of PBMCs obtained from about 10 to 50 ml of bone marrow aspirate is about 5×10 7 ~about 10×10 7 PBMCs. In another embodiment, the number of PMBCs obtained is about 7×10 7 PBMCs.

[0149]

[0200] In one embodiment of the present invention, about 0.5×10 7 ~about 1.5×10 7 expansion culture starting cell materials are obtained from about 5×10 6 ~about 10×10 6 PBMCs. In one embodiment of the present invention, about 1×10 6 expansion culture starting cell materials are obtained.

[0150]

[0201] In one embodiment of the present invention, the total number of MILs recovered at the end of the expansion culture period is about 0.01×10 9 cells to about 1×10 9 cells, about 0.05×10 9 cells to about 0.9×109 cells, about 0.1×10 9 cells to about 0.85×10 9 cells, about 0.15×10 9 cells to about 0.7×10 9 cells, about 0.2×10 9 cells to about 0.65×10 9 cells, about 0.25×10 9 cells to about 0.6×10 9 cells, about 0.3×10 9 cells to about 0.55×10 9 cells, about 0.35×10 9 cells to about 0.5×10 9 cells or about 0.4×10 9 cells to about 0.45×10 9 cells.

[0151]

[0202] In one embodiment of the present invention, 12×10 6 cells of PBMC derived from bone marrow aspirate generate approximately 1.4×10 5 cells of starting cell material, which generates approximately 1.1×10 7 cells of MIL at the end of the expansion culture process.

[0152]

[0203] In one embodiment of the present invention, the MIL expanded from bone marrow PBMC using the above MIL method 3 contains a higher percentage of CD8+ cells and a lower number of LAG3+ and PD1+ cells compared to the MIL expanded using MIL method 1 or MIL method 2. In one embodiment of the present invention, the PBL expanded from blood PBMC using the above MIL method 3 contains a higher percentage of CD8+ cells and increased levels of IFNγ production compared to the PBL expanded using MIL method 1 or MIL method 2.

[0153]

[0204] In one embodiment of the present invention, the clinical dose of MIL useful for patients with acute myeloid leukemia (AML) is in the range of about 4×10 8 to about 2.5×10 9 cells of MIL. In another embodiment, the number of MIL provided in the pharmaceutical composition of the present invention is 9.5×10 8are individual MILs. In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 4.1×10 8 In another embodiment, the number of MILs provided in the pharmaceutical composition of the present invention is 2.2×10 9 .

[0154]

[0205] In any of the foregoing embodiments, the PBMCs can be obtained from whole blood samples, from bone marrow, by apheresis, from buffy coats, or from any other method known in the art for obtaining PBMCs.

[0155] Method for Expanding and Culturing TIL Using the "2A Process"

[0206] In one embodiment of the present invention, the present invention provides an apparatus and method for expanding and culturing T cells derived from bone marrow and / or peripheral blood. In one embodiment of the present invention, the T cells are polyclonal but have enhanced tumor specificity from the bone marrow microenvironment in a highly tumor-specific manner. In one embodiment, the bone marrow microenvironment is used to maintain and expand the culture of T cells. In one embodiment of the present invention, there is an expansion culture of approximately 25 to 100 times that of TIL in a 7-day or 14-day expansion culture process. In one embodiment, the expansion multiple of TIL is about 30 to 90 times. In one embodiment, the expansion multiple is about 35 to 85 times. In one embodiment, the expansion multiple is about 40 to 80 times. In one embodiment, the expansion multiple is about 45 to 75 times. In another embodiment, the expansion multiple is about 40 to 70 times. In another embodiment, the expansion multiple is about 45 to 65 times. In another embodiment, the expansion multiple is about 25 times, about 30 times, about 35 times, about 40 times, about 45 times and 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times or about 100 times.

[0156]

[0207] In one embodiment of the present invention, the T cell manufacturing process does not require an intervention for selecting tumor specificity. In one embodiment of the present invention, the T cell manufacturing process does not require the presence of a tumor in the bone marrow and / or peripheral blood during T cell expansion culture. In one embodiment, the T cells are expanded in culture in the presence of substantially complete bone marrow.

[0157]

[0208] In one embodiment, the present invention provides a method for extracting T cells from bone marrow and / or peripheral blood as described in Example 21, in particular, in International Publication No. WO 2010 / 062742, which is incorporated herein by reference. In one embodiment, the present invention provides a method for extracting T cells from bone marrow and / or peripheral blood, for example, as described in Noonan, et al., 2005, Cancer Res. 65:2026-2034, which is incorporated herein by reference.

[0158]

[0209] In one embodiment, methods for obtaining bone marrow and / or peripheral blood known to those skilled in the art are useful in the present invention. In one embodiment of the present invention, the bone marrow and / or peripheral blood are obtained using needle aspiration. In one embodiment of the present invention, bone marrow from a patient is aspirated into a heparin-containing syringe and stored overnight at room temperature. In one embodiment of the present invention, after storage, the contents of the syringe are pooled into a sterile container and tested for quality. The bone marrow is centrifuged using lymphocyte separation medium (LSM) and COBE Spectra to concentrate mononuclear cells (MNC). The cells within the gradient are collected up to red blood cells and washed using HBSS. The MNC are cryopreserved using a hetastarch-based cryoprotectant supplemented with 2% HSA and 5% DMSO, and a portion of the MNC is reserved for quality control. The QC vial is thawed to measure the cell content of CD3+ and CD38 + / 138 + in the MNC product. It is important to note that the collection of bone marrow does not limit the present invention.

[0159]

[0210] In one embodiment of the present invention, bone marrow is aspirated and fractionated by a lymphocyte separation medium density gradient, and the cells are collected to approximately the level of the erythrocyte pellet. In one embodiment, this fractionation method substantially removes erythrocytes and neutrophils and provides substantially complete bone marrow. In one embodiment, the resulting fractionated material is T cells and tumor cells. In one embodiment of the present invention, the method can be carried out without a T cell-specific separation step and without a tumor cell separation step, for example, without labeling T cells with an antibody or other cell type-specific detectable label and without sorting using fluorescence-activated cell sorting (FACS).

[0160]

[0211] In one embodiment of the present invention, the obtained bone marrow is ficolled or peripheral blood is suspended in AIM-V medium at 200 μL / well at 1×10 6 cells / mL under serum-free conditions.

[0161]

[0212] In one embodiment of the present invention, bone marrow is collected from a subject who is not in complete remission. In one embodiment of the present invention, bone marrow is collected from a subject who is in complete remission.

[0162]

[0213] In one embodiment of the present invention, bone marrow can be obtained and frozen. In one embodiment, bone marrow can be obtained and immediately used to extract T cells.

[0163]

[0214] In a further embodiment and according to any of the above, the present invention provides a method for expanding TIL, comprising contacting a population of TIL comprising at least one TIL obtained from a liquid tumor. All discussions herein regarding the expansion culture of TIL are applicable to the expansion culture of TIL obtained from hematological malignancies including bone marrow, peripheral blood, and / or liquid tumors.

[0164]

[0215] In one embodiment, the present invention is a process for preparing a population of tumor-infiltrating lymphocytes (TIL) from a tumor, comprising (a) contacting the fragmented tumor with a first cell culture medium; (b) A step of performing an initial expansion culture (pre-REP) of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least five times more numerous than the first population of TILs, and the first cell culture medium contains IL-2; (c) A step of performing a second expansion culture of the second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50 times more numerous than the second population of TILs 7 days after the start of the second expansion culture, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the second expansion culture is performed over a period of 14 days or less; (d) A step of recovering the third population of TILs including, wherein the tumor is a liquid tumor and the cancer is a hematological malignancy, a process is provided.

[0165]

[0216] In one embodiment, the present invention is a process for expanding a population of TILs, comprising a first pre-rapid expansion culture (pre-REP) process, followed by a second expansion culture process (which may be a rapid expansion culture process - REP), wherein the cell culture medium used for the expansion culture contains IL-2 at a concentration selected from the group consisting of 100 IU / mL to 10,000 IU / mL, 200 IU / mL to 5,000 IU / mL, 300 IU / mL to 4,800 IU / mL, 400 IU / mL to 4,600 IU / mL, 500 IU / mL to 4,400 IU / mL, 600 IU / mL to 4,200 IU / mL, 700 IU / mL to 4,000 IU / mL, 800 IU / mL to 3,800 IU / mL, 900 IU / mL to 3,600 IU / mL, 1,000 IU / mL to 3,400 IU / mL, 1,100 IU / mL to 3,200 IU / mL, 1,200 IU / mL to 3,000 IU / mL, 1,300 IU / mL to 2,800 IU / mL, 1,400 IU / mL to 2,600 IU / mL, 1,500 IU / mL to 2,400 IU / mL, 1,600 IU / mL to 2,200 IU / mL, 1,700 IU / mL to 2,000 IU / mL, 5,500 IU / mL to 9,500 IU / mL, 6,000 IU / mL to 9,000 IU / mL, 6500 IU / mL to 8,500 IU / mL, 7,000 IU / mL to 8,000 IU / mL, and 7,500 IU / mL to 8,000 IU / mL.

[0166]

[0217] In one embodiment, the present invention provides a process for expanding a population of TILs, which includes a pre-rapid expansion culture (pre-REP) process and a rapid expansion culture process (REP). The cell culture medium used for the expansion culture contains IL-2 at a concentration selected from the group consisting of about 100 IU / mL, about 200 IU / mL, about 300 IU / mL, about 400 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 100 IU / mL, about 500 IU / mL, about 600 IU / mL, about 700 IU / mL, about 800 IU / mL, about 900 IU / mL, about 1,000 IU / mL, about 1,100 IU / mL, about 1,200 IU / mL, about 1,300 IU / mL, about 1,400 IU / mL, about 1,500 IU / mL, about 1,600 IU / mL, about 1,700 IU / mL, about 1,800 IU / mL, about 1,900 IU / mL, about 2,000 IU / mL, about 2,100 IU / mL, about 2,200 IU / mL, about 2,300 IU / mL, about 2,400 IU / mL, about 2,500 IU / mL, about 2,600 IU / mL, about 2,700 IU / mL, about 2,800 IU / mL, about 2,900 IU / mL, about 3,000 IU / mL, about 3,100 IU / mL, about 3,200 IU / mL, about 3,300 IU / mL, about 3,400 IU / mL, about 3,500 IU / mL, about 3,600 IU / mL, about 3,700 IU / mL, about 3,800 IU / mL, about 3,900 IU / mL, about 4,000 IU / mL, about 4,100 IU / mL, about 4,200 IU / mL, about 4,300 IU / mL, about 4,400 IU / mL, about 4,500 IU / mL, about 4,600 IU / mL, about 4,700 IU / mL, about 4,800 IU / mL, about 4,900 IU / mL, about 5,000 IU / mL, about 5,100 IU / mL, about 5,200 IU / mL, about 5,300 IU / mL, about 5,400 IU / mL, about 5,500 IU / mL, about 5,600 IU / mL, about 5,700 IU / mL, about 5,800 IU / mL, about 5,900 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, about 8,000 IU / mL, about 8,500 IU / mL, about 9,000 IU / mL, about 9,500 IU / mL and about 10,000 IU / mL.

[0167]

[0218] In one embodiment, the present invention provides a process for expanding a population of TILs, including a pre-rapid expansion culture (pre-REP) process. In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising the step of contacting a population of TILs obtained from a liquid tumor with a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of 1000 IU / mL to 6000 IU / mL.

[0168]

[0219] In one embodiment, the present invention is a pre-REP process for expanding a population of TILs, comprising the step of contacting a population of TILs obtained from a liquid tumor with a cell culture medium, the cell culture medium further comprising IL-2 at an initial concentration of about 6000 IU / mL.

[0169]

[0220] In one embodiment, the REP can be performed in a gas-permeable container using TIL obtained from a liquid tumor according to the present disclosure by any suitable method. For example, TIL can be rapidly expanded in culture using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulation can include, for example, about 30 ng / mL of OKT-3, a monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). TIL can optionally be further stimulated in vitro to rapidly expand in culture using one or more antigens, such as an antigenic portion thereof, such as a cancer epitope, that can be expressed from a vector such as 0.3 μM MART-1:26-35 (27L) or gpl00:209-217 (210M), in the presence of a T cell growth factor such as 300 IU / mL of IL-2 or IL-15, optionally. Other suitable antigens can include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TIL can also be rapidly expanded in culture by restimulation with the same cancer antigen pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TIL can be further restimulated, for example, by irradiated autologous lymphocytes, or by irradiated HLA-A2+ allogeneic lymphocytes and IL-2, by way of example.

[0170]

[0221] In one embodiment, a method for expanding TIL may include using from about 5000 mL to about 25000 mL of cell culture medium, from about 5000 mL to about 10000 mL of cell culture medium, or from about 5800 mL to about 8700 mL of cell culture medium. In one embodiment, a method for expanding TIL may include using from about 1000 mL to about 2000 mL of cell culture medium, from about 2000 mL to about 3000 mL of cell culture medium, from about 3000 mL to about 4000 mL of cell culture medium, from about 4000 mL to about 5000 mL of cell culture medium, from about 5000 mL to about 6000 mL of cell culture medium, from about 6000 mL to about 7000 mL of cell culture medium, from about 7000 mL to about 8000 mL of cell culture medium, from about 8000 mL to about 9000 mL of cell culture medium, from about 9000 mL to about 10000 mL of cell culture medium, from about 10000 mL to about 15000 mL of cell culture medium, from about 15000 mL to about 20000 mL of cell culture medium, or from about 20000 mL to about 25000 mL of cell culture medium. In one embodiment, to expand the number of TIL, one or fewer types of cell culture medium are used. Any suitable cell culture medium, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA) can be used. In this regard, the method of the present invention advantageously reduces the amount of medium and the number of types of medium required to expand the number of TIL. In one embodiment, expanding the number of TIL may include feeding the cells at a frequency of once every 3 or 4 days or less. Increasing the number of cells in a gas-permeable container simplifies the procedure required to expand the number of cells by reducing the feeding frequency required to expand the cells in culture.

[0171]

[0222] In one embodiment, the second expansion culture is performed using a gas-permeable container. Such an embodiment is where the cell population is from about 5×10 5 cells / cm 2 to 10×10 6 ~30×10 6 cells / cm 2It enables expansion culture. In one embodiment, this expansion culture occurs without feeding. In one embodiment, this expansion culture occurs without feeding as long as the medium is at a height of about 10 cm in a gas-permeable flask. In one embodiment, this is without feeding but involves the addition of one or more cytokines. In one embodiment, the cytokine can be added as a bolus without the need to mix it with the medium. Such containers, devices, and methods are known in the art and have been used for the expansion culture of TILs, including those described in U.S. Patent Application Publication No. 2014 / 0377739A1, International Publication No. 2014 / 210036A1, U.S. Patent Application Publication No. 2013 / 0115617A1, International Publication No. 2013 / 188427A1, U.S. Patent Application Publication No. 2011 / 0136228A1, U.S. Patent No. 8,809,050, International Publication No. 2011 / 072088A2, U.S. Patent Application Publication No. 2016 / 0208216A1, U.S. Patent Application Publication No. 2012 / 0244133A1, International Publication No. 2012 / 129201A1, U.S. Patent Application Publication No. 2013 / 0102075A1, U.S. Patent No. 8,956,860, International Publication No. 2013 / 173835A1, and U.S. Patent Application Publication No. 2015 / 0175966A1, the disclosures of which are incorporated herein by reference. Such a process is also described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated herein by reference.

[0172]

[0223] In one embodiment, the gas-permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container is 10 cm 2 in gas-permeable culture surface. In one embodiment, the gas-permeable container contains a cell culture medium volume of 40 mL. In one embodiment, the gas-permeable container provides 100 million to 300 million TILs after two medium exchanges.

[0173]

[0224] In one embodiment, the gas-permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container includes a gas-permeable culture surface of 100 cm 2 . In one embodiment, the gas-permeable container includes a cell culture medium volume of 450 mL. In one embodiment, the gas-permeable container provides 1 billion to 3 billion TILs after two medium exchanges.

[0174]

[0225] In one embodiment, the gas-permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container includes a gas-permeable culture surface of 100 cm 2 . In one embodiment, the gas-permeable container includes a cell culture medium volume of 1000 mL. In one embodiment, the gas-permeable container provides 1 billion to 3 billion TILs without medium exchange.

[0175]

[0226] In one embodiment, the gas-permeable container is a G-Rex 100L flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container includes a gas-permeable culture surface of 100 cm 2 . In one embodiment, the gas-permeable container includes a cell culture medium volume of 2000 mL. In one embodiment, the gas-permeable container provides 1 billion to 3 billion TILs without medium exchange.

[0176]

[0227] In one embodiment, the gas-permeable container is a G-Rex 24-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container includes a plate having wells, and each well has a 2 cm 2It includes a gas-permeable culture surface. In one embodiment, the gas-permeable container includes a plate with wells, and each well contains a cell culture medium volume of 8 mL. In one embodiment, the gas-permeable container provides 20 million to 60 million cells per well after two medium exchanges.

[0177]

[0228] In one embodiment, the gas-permeable container is a G-Rex 6-well plate (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In one embodiment, the gas-permeable container includes a plate with wells, and each well has a gas-permeable culture surface of 10 cm 2 It includes. In one embodiment, the gas-permeable container includes a plate with wells, and each well contains a cell culture medium volume of 40 mL. In one embodiment, the gas-permeable container provides 100 million to 300 million cells per well after two medium exchanges.

[0178]

[0229] In one embodiment, the cell culture medium in the first and / or second gas-permeable containers is not filtered. The use of unfiltered cell culture medium can simplify the procedures necessary to expand the cell number. In one embodiment, the cell culture medium in the first and / or second gas-permeable containers lacks beta-mercaptoethanol (BME).

[0179]

[0230] In one embodiment, it includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium; obtaining TIL from the tumor tissue sample; expanding the number of TIL in a second gas-permeable container containing cell culture medium for about 14 to about 42 days, for example about 28 days, which is the required period of this method.

[0180]

[0231] In one embodiment, the cell culture medium contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU / mL of IL-2. In one embodiment, 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. In one embodiment, the cell culture medium contains IL-2 at a concentration of 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.

[0181]

[0232] In one embodiment, the cell culture medium contains the OKT-3 antibody. In a preferred embodiment, the cell culture medium contains about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL and about 1 μg / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains the OKT-3 antibody at a concentration of 0.1 ng / mL - 1 ng / mL, 1 ng / mL - 5 ng / mL, 5 ng / mL - 10 ng / mL, 10 ng / mL - 20 ng / mL, 20 ng / mL - 30 ng / mL, 30 ng / mL - 40 ng / mL, 40 ng / mL - 50 ng / mL and 50 ng / mL - 100 ng / mL.

[0182]

[0233] In one embodiment, TILs are expanded in a gas-permeable container. Gas-permeable containers are 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 / 0106717A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are expanded in a gas-permeable bag. In one embodiment, TILs are expanded using a cell expansion culture system that expands TILs in a gas-permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are expanded using a cell expansion culture system that expands TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion culture system includes 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, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L. In one embodiment, the cell expansion culture system includes a gas-permeable cell bag having a volume in the range selected from the group consisting of 50 - 150 mL, 150 - 250 mL, 250 - 350 mL, 350 - 450 mL, 450 - 550 mL, 550 - 650 mL, 650 - 750 mL, 750 - 850 mL, 850 - 950 mL, and 950 - 1050 mL. In one embodiment, the cell expansion culture system includes a gas-permeable cell bag having a volume in the range selected from the group consisting of 1 L - 2 L, 2 L - 3 L, 3 L - 4 L, 4 L - 5 L, 5 L - 6 L, 6 L - 7 L, 7 L - 8 L, 8 L - 9 L, 9 L - 10 L, 10 L - 11 L, 11 L - 12 L, 12 L - 13 L, 13 L - 14 L, 14 L - 15 L, 15 L - 16 L, 16 L - 17 L, 17 L - 18 L, 18 L - 19 L, and 19 L - 20 L.In one embodiment, the cell expansion culture system includes a gas-permeable cell bag having a volume in a range selected from the group consisting of 0.5 L to 5 L, 5 L to 10 L, 10 L to 15 L, 15 L to 20 L, 20 L to 25 L, and 25 L to 30 L. In one embodiment, the cell expansion culture system uses a rocking time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days. In one embodiment, the cell expansion culture system uses a rocking time of 30 minutes to 1 hour, 1 hour to 12 hours, 12 hours to 1 day, 1 day to 7 days, 7 days to 14 days, 14 days to 21 days, and 21 days to 28 days. In one embodiment, the cell expansion culture system uses a rocking speed of about 2 rockings per minute, about 5 rockings per minute, about 10 rockings per minute, about 20 rockings per minute, about 30 rockings per minute, and about 40 rockings per minute. In one embodiment, the cell expansion culture system uses a rocking speed of 2 rockings per minute to 5 rockings per minute, 5 rockings per minute to 10 rockings per minute, 10 rockings per minute to 20 rockings per minute, 20 rockings per minute to 30 rockings per minute, and 30 rockings per minute to 40 rockings per minute. In one embodiment, the cell expansion culture system uses a rocking angle of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In one embodiment, the cell expansion culture system uses a rocking angle of 2° to 3°, 3° to 4°, 4° to 5°, 5° to 6°, 6° to 7°, 7° to 8°, 8° to 9°, 9° to 10°, 10° to 11°, and 11° to 12°.

[0183]

[0234] In one embodiment, a method of expanding and culturing TIL obtained from a liquid tumor further includes a step of selecting TIL for excellent tumor reactivity. Any selection method known in the art can be used. For example, the method described in US Patent Application Publication No. 2016 / 0010058A1 (this disclosure is incorporated herein by reference) can be used for the selection of TIL for excellent tumor responsiveness.

[0184]

[0235] In one embodiment, the present invention provides a method for expanding a population of TILs from a liquid tumor, the method including steps as described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated herein by reference. For example, a tumor or a part thereof can be placed in an enzyme medium and mechanically separated for approximately 1 minute. The mixture can then be incubated at 37° C. in 5% CO2 for 30 minutes and then mechanically disrupted again for approximately 1 minute. After incubation at 37° C. in 5% CO2 for 30 minutes, the tumor or a part thereof can be mechanically disrupted for the third time for approximately 1 minute. After the third mechanical disruption, if large tissue pieces are present, one or two additional mechanical separations can be applied to the sample, with or without an additional 30-minute incubation at 37° C. in 5% CO2. At the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells. TIL culture is initiated in 24-well plates (Costar 24-well cell culture clusters, flat bottom; Corning Incorporated, Corning, NY), and each well is seeded with 1×10 6 tumor digestion cells or one tumor fragment of approximately 1 to 8 mm 3 in size. The CM contains Roswell Park Memorial Institute (RPMI) 1640 buffer containing GlutaMAX, to which 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin are added. The culture can be initiated in gas-permeable flasks (G-Rex 10; Wilson Wolf Manufacturing, New Brighton) having a 40 mL volume and a 10 cm 2 gas-permeable silicon bottom, and each flask is seeded with 10 to 40×10 6It can be filled with viable tumor digesting cells or 5 - 30 tumor fragments. G-Rex 10 and 24-well plates can be incubated in a humidified incubator at 37°C in 5% CO2. Five days after the start of culture, half of the medium can be removed and replaced with fresh CM and IL-2. Five days later, half of the medium can be replaced every 2 - 3 days. Using TIL obtained from the liquid tumors of the present disclosure, as described elsewhere herein, a second expansion culture protocol (REP) of TIL can be performed using T-175 flasks and gas-permeable bags or gas-permeable G-Rex flasks. In the REP in T-175 flasks, 1×10 6 TIL can be suspended in 150 mL of medium in each flask. TIL can be cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3 antibody (OKT-3). T-175 flasks can be incubated at 37°C in 5% CO2. Half of the medium can be replaced on day 5 with 50 / 50 medium containing 3000 IU / mL of IL-2. On day 7, cells from two T-175 flasks can be mixed in a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 can be added to 300 mL of the TIL suspension. The cell number in each bag can be counted daily or every other day, and fresh medium can be added to maintain the cell number at 0.5 - 2.0×10 6 cells / mL. In the REP in a 500 mL capacity flask (e.g., G-Rex 100, Wilson Wolf Manufacturing) with a 100 cm 2 gas-permeable silicon bottom as described elsewhere herein, 5×10 6 or 10×10 6Individual TILs can be cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3 antibody (OKT-3). G-Rex 100 flasks can be incubated at 37 °C under 5% CO2. On day 5, 250 mL of the supernatant can be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. The obtained TIL pellet can be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL of IL-2 and returned to and added to the G-Rex 100 flask. When continuously expanding TILs in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 are suspended in 300 mL of the medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-Rex 100 flasks. Then, approximately 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 can be added to each flask. Then, the G-Rex 100 flasks are incubated at 37 °C in 5% CO2, and 4 days later, 150 mL of AIM-V with 3000 IU / mL of IL-2 can be added to each G-Rex 100 flask. Thereafter, REP can be completed by harvesting the cells on day 14 of culture.

[0185]

[0236] In one embodiment, a method of expanding or treating cancer comprises the step of obtaining TILs from a patient's tumor sample. The patient's tumor sample can be obtained using methods known in the art. For example, TILs can be obtained from enzymatic tumor digest and tumor fragments (sized approximately 1 to approximately 8 mm 3) can be cultured from. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL of DNase, and 1.0 mg / mL of collagenase), followed by mechanical separation (e.g., using a tissue dissociator). The tumor digest can be prepared by placing the tumor in the enzyme medium, mechanically separating the tumor for approximately 1 minute, then incubating at 37°C under 5% CO2 for 30 minutes, and then repeating the cycle of mechanical separation and incubation under the aforementioned conditions until only very small tissue pieces are present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference, can be used. Any of the aforementioned methods can be used in any of the embodiments described herein for a method of expanding TIL or a method of treating cancer.

[0186]

[0237] In one embodiment, as described above, a second / REP expansion culture process of TIL can be carried out using a T-175 flask and a gas-permeable bag (Tran, et al, J. Immunother. 2008, 31, 742-51; Dudley, et al, J. Immunother. 2003, 26, 332-42) or using a gas-permeable culture device (G-Rex flask, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In the TIL expansion culture in a T-175 flask, 1×10 suspended in 150 mL of medium 6Individual TILs can be added to each T-175 flask. The TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU (International Units) of IL-2 per mL and 30 ng of anti-CD3 antibody (e.g., OKT-3) per mL. The T-175 flasks can be incubated at 37 °C in 5% CO2. Half of the medium can be replaced on day 5 with 50 / 50 medium containing 3000 IU of IL-2 per mL. On day 7, the cells from two T-175 flasks are mixed in a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU of IL-2 per mL are added to the 300 mL TIL suspension. The cell count in each bag is counted daily or every other day, and fresh medium is added to maintain the cell count at 0.5 - 2.0×10 6 cells / mL.

[0187]

[0238] In one embodiment, in a second / REP TIL expansion culture in a 500 mL capacity gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) having a 100 cm 2 gas-permeable silicon bottom, 5×10 6 or 10×10 6Individual TILs can be cultured in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU of IL-2 per mL, and 30 ng of anti-CD3 (OKT-3) per mL. G-Rex 100 flasks can be incubated at 37°C under 5% CO2. On day 5, 250 mL of the supernatant can be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (revolutions per minute; 491×g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU of IL-2 per mL and returned to and added to the original G-Rex 100 flask. When TILs are continuously expanded in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 flask can be suspended in 300 mL of the medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots to be used for seeding three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU of IL-2 per mL can be added to each flask. The G-Rex 100 flasks can be incubated at 37°C in 5% CO2, and after 4 days, 150 mL of AIM-V with 3000 IU of IL-2 per mL can be added to each G-Rex 100 flask. The cells can be harvested on day 14 of culture.

[0188]

[0239] In one embodiment, TILs can be prepared as follows. 2 mm 3The tumor fragments are cultured in complete medium (CM) containing AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc., Manassas, VA), 100 U / mL penicillin (Invitrogen Life Technologies), 100 μg / mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc., Winchester, VA), and 600 IU / mL rhIL-2 (Chiron, Emeryville, CA). For enzymatic digestion of liquid tumors, the tumor specimens are minced and placed into RPMI-1640, washed, centrifuged at 800 rpm for 5 minutes at 15 - 22 °C, resuspended in enzymatic digestion buffer (0.2 mg / mL collagenase and 30 units / ml DNase in RPMI-1640), and subsequently rotated overnight at room temperature. TILs generated from the fragments are grown in CM for 3 - 4 weeks, either freshly expanded or cryopreserved in heat-inactivated HAB serum containing 10% dimethyl sulfoxide (DMSO) and stored at -180 °C until the time of study. Tumor-associated lymphocytes (TALs) obtained from ascites collection are seeded at 3×10 6 cells / well in 24-well plates in CM. Using a low-power inverted microscope, the growth of TILs is examined every other day.

[0189] Exemplary embodiments of the TIL manufacturing process (“2A process”)

[0169] An exemplary TIL manufacturing / expansion process known as Process 2A is schematically shown in FIG. 22. In certain embodiments, the method produces TILs that can increase the replication cycle upon administration to a subject / patient and thus may provide additional therapeutic benefits beyond mature TILs (i.e., TILs that have undergone more replications prior to administration to the subject / patient). The characteristics of young TILs have been described in the literature. For example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010), Huang et al., J Immunother, 28(3):258-267 (2005), Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013), Besser et al., J Immunother 32:415-423 (2009), Robbins, et al., J Immunol 2004;173:7125-7130, Shen et al., J Immunother, 30:123-129 (2007), Zhou, et al., J Immunother, 28:53-62 (2005) and Tran, et al., J Immunother, 31:742-751 (2008), all of which are hereby incorporated by reference in their entirety.

[0190]

[0170] As discussed herein, the present invention may include steps related to increasing the metabolic activity, and thus relative health, of cryopreserved TILs by restimulating them prior to transplantation into a patient, and a method of testing for said metabolic health. Generally as outlined herein, TILs are generally harvested from a patient sample and manipulated to expand their numbers prior to transplantation into a patient. In some embodiments, the TILs may optionally be genetically engineered as discussed below.

[0191]

[0171] In some embodiments, TILs can be cryopreserved. After thawing, the TILs can be restimulated to enhance their metabolism prior to infusion into the patient.

[0192]

[0172] In some embodiments, as detailed below and in the Examples and Figures, the first expansion culture (including the process referred to as pre-REP) is shortened to 7 - 14 days compared to conventional expansion culture methods, and the second expansion culture (including the process referred to as REP) is shortened to 7 - 14 days.

[0193]

[0173] Figure 23 shows an exemplary 2A process. As shown in Figure 23 and described in further detail below, in some embodiments, the first expansion culture (step B) is shortened to 11 days, and the second expansion culture (step D) is shortened to 11 days. In some embodiments, as detailed herein, the combination of the first and second expansion cultures (steps B and D) is shortened to 22 days. As will be appreciated, the process shown in Figure 23 and described below is exemplary, and the methods described herein encompass changes to and additions of the described steps and any combinations thereof. Exemplary embodiments of this process are described in PCT Application No. PCT / US Patent Application Publication No. 2018 / 012633, which is hereby incorporated by reference in its entirety.

[0194] A. Step A: Obtaining a patient tumor sample

[0174] Generally, TILs are first obtained from a patient tumor sample ("primary TILs"), then expanded to a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally phenotyped and metabolic parameters are determined as indicators of TIL health.

[0195]

[0175] Patient tumor samples can generally be obtained by means of surgical resection, needle biopsy, apheresis or other means for obtaining samples containing a mixture of tumor and TIL cells, using methods known in the art. Generally, tumor samples can be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. Tumor samples can also be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be of any cancer type, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma and melanoma). In some embodiments, useful TILs are obtained from melanoma tumors, particularly as they have been reported to have particularly high levels of TILs. In some embodiments, the tumor is larger than about 1.5 cm and smaller than about 4 cm. In some embodiments, the tumor is smaller than 4 cm.

[0196]

[0176] After obtaining, the tumor samples are generally fragmented into pieces of 1 to about 8 mm using sharp dissection 3 and about 2 to 3 mm 3is particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL of DNase, and 1.0 mg / mL of collagenase), followed by mechanical dissociation (e.g., using a tissue dissociator). The tumor digest is prepared by placing the tumor in the enzyme medium, mechanically dissociating the tumor for about 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only very small tissue pieces remain. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference, can be used. Any of the foregoing methods can be used in any of the embodiments described herein for the method of expanding TILs or for the method of treating cancer.

[0197]

[0177] Generally, the collected cell suspension is referred to as a "primary cell population" or a "freshly collected" cell population.

[0198]

[0178] In one embodiment, TILs can be cultured initially from an enzymatic tumor digest and tumor fragments obtained from a patient.

[0199]

[0179] In some embodiments, TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 1 mm 3 ~10 mm 3 in size. In some embodiments, the tumor fragments are about 1 mm 3 ~8 mm 3 in size. In some embodiments, the tumor fragments are about 1 mm 3It is. In some embodiments, the tumor fragment is about 2 mm 3 It is. In some embodiments, the tumor fragment is about 3 mm 3 It is. In some embodiments, the tumor fragment is about 4 mm 3 It is. In some embodiments, the tumor fragment is about 5 mm 3 It is. In some embodiments, the tumor fragment is about 6 mm 3 It is. In some embodiments, the tumor fragment is about 7 mm 3 It is. In some embodiments, the tumor fragment is about 8 mm 3 It is. In some embodiments, the tumor fragment is about 9 mm 3 It is. In some embodiments, the tumor fragment is about 10 mm 3 It is. In some embodiments, the tumor fragment is about 8 - 27 mm 3 It is. In some embodiments, the tumor fragment is about 10 - 25 mm 3 It is. In some embodiments, the tumor fragment is about 15 - 25 mm 3 It is. In some embodiments, the tumor fragment is about 8 - 20 mm 3 It is. In some embodiments, the tumor fragment is about 15 - 20 mm 3 It is. In some embodiments, the tumor fragment is about 8 - 15 mm 3 It is. In some embodiments, the tumor fragment is about 8 - 10 mm 3 It is.

[0200]

[0180] In some embodiments, the number of tumor fragments is about 40 to about 50 tumor fragments. In some embodiments, the number of tumor fragments is about 40 tumor fragments. In some embodiments, the number of tumor fragments is about 50 tumor fragments. In some embodiments, the tumor fragment size is about 8 - 27 mm 3 and there are less than about 50 tumor fragments.

[0201]

[0181] In some embodiments, TILs are obtained from tumor digests. In some embodiments, the tumor digest is created by incubation in an enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be incubated at 37 °C under 5% CO2 for 30 minutes, and then it can be mechanically disrupted again for about 1 minute. After incubating again at 37 °C under 5% CO2 for 30 minutes, the tumor can be mechanically disrupted a third time for about 1 minute. In some embodiments, if large tissue pieces are present after the third mechanical disruption, one or two additional mechanical dissociations with or without an additional 30-minute incubation at 37 °C under 5% CO2 are applied to the sample. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll can be performed to remove such cells.

[0202] B. Step B: First expansion culture

[0182] After detachment or digestion of the tumor fragments in Step A, the resulting cells are cultured in serum-containing IL-2 under conditions favorable for the growth of TILs over tumors and other cells. In some embodiments, the tumor digest is incubated in a medium containing inactivated human AB serum with 6000 IU / mL of IL-2 in 2 mL wells. This primary cell population is cultured over a period of several days, generally 3 - 14 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, this primary cell population is cultured over a period of 7 - 14 days, thereby obtaining a bulk TIL population, generally about 1×10 8 individual bulk TIL cells. In some embodiments, this primary cell population is cultured over a period of 10 - 14 days, thereby obtaining a bulk TIL population, generally about 1×108 Individual bulk TIL cells are obtained. In some embodiments, this primary cell population is cultured over a period of about 11 days, thereby resulting in a bulk TIL population, generally about 1×10 8 Individual bulk TIL cells are obtained. In some embodiments, this primary cell population is cultured over a period of about 11 days, thereby resulting in a bulk TIL population, generally about 200×10 6 Individual bulk TIL cells or fewer are obtained.

[0203]

[0183] In preferred embodiments, as described below and herein, an initial bulk TIL expansion culture step (e.g., step B shown in FIG. 23 which may include a process referred to as pre-REP), followed by a subsequent second expansion culture (step D, including a process referred to as a rapid expansion protocol (REP) step) as described below under step D and herein, followed by optional cryopreservation and a subsequent second step D (including a process referred to as a restimulation REP step) can be used to expand the TIL. The TIL obtained by this process can optionally be characterized by phenotypic features and metabolic parameters as described herein.

[0204]

[0184] In an embodiment, when TIL culture is initiated in a 24-well plate using, for example, a Costar 24-well cell culture cluster, flat bottom (Corning Incorporated, Corning, NY), each well can be seeded with 1×10 6 tumor digest cells or 1 tumor fragment in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is about 1 mm 3 ~10 mm 3 in size.

[0205]

[0185] In one embodiment, the CM of step B consists of GlutaMAX-containing RPMI 1640 supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg / mL gentamicin. The culture is in a 40 mL volume and 10 cm 2 In embodiments where the culture is initiated in a gas-permeable flask with a gas-permeable silicon bottom (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask is loaded with 10 - 40 × 10 6 viable cells of tumor digest or 5 - 30 tumor fragments in 10 - 40 mL of IL-2-containing CM. Both G-Rex10 and 24-well plates are incubated at 37 °C under 5% CO2 in a humidified incubator. Half of the medium is removed 5 days after the start of the culture and replenished with fresh CM and IL-2, and half of the medium is exchanged every 2 - 3 days after day 5.

[0206]

[0186] In one embodiment, the cell culture medium further contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU / mL of IL-2. In one embodiment, 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. In one embodiment, the cell culture medium contains 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.

[0207]

[0187] In some embodiments, as discussed in the examples and figures, the first expansion culture (including the process referred to as pre-REP; step B) process is shortened to 3 to 14 days. In some embodiments, as discussed in the examples and as shown in FIGS. 4 and 5, the first expansion culture of step B is shortened to 7 to 14 days. In some embodiments, as discussed in the examples, the first expansion culture of step B is shortened to 10 to 14 days. In some embodiments, as discussed in the examples, the first expansion culture of step B is shortened to 11 days.

[0208]

[0188] In some embodiments, IL-2, IL-7, IL-15, and IL-21 and combinations thereof may be included during the step B process, as described herein.

[0209]

[0189] In some embodiments, step B is performed in a closed-system bioreactor. In some embodiments, a closed system is used for TIL expansion culture, as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100.

[0210] C. Step C: Transition from the first expansion culture to the second expansion culture

[0190] In some embodiments, the bulk TIL population from step B can be cryopreserved immediately using methods known in the art and described herein. Alternatively, the bulk TIL population can be subjected to a second expansion culture (REP), as discussed below, and then cryopreserved.

[0211]

[0191] In some embodiments, the TIL of step B is not stored and the TIL of step B proceeds directly to step D. In some embodiments, as further described herein, the transition is performed in a closed system.

[0212] D. Step D: Second expansion culture

[0192] In some embodiments, the TIL cell population is expanded in number after harvesting and initial bulk processing (i.e., after steps A and B). This may include an expansion culture process that is generally referred to herein as a second expansion culture and is generally referred to in the art as a rapid expansion culture process (REP). The second expansion culture can generally be achieved using a culture medium in a gas-permeable container that contains several components, including feeder cells, a cytokine source, and an anti-CD3 antibody. In some embodiments, the second expansion culture may include scaling up to increase the number of TILs obtained in the second expansion culture.

[0213]

[0193] In one embodiment, REP and / or the second expansion culture can be performed in a gas-permeable container using the methods of the present disclosure. For example, TIL can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Examples of non-specific T cell receptor stimulation include, for example, about 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). TIL can optionally be further stimulated in vitro by one or more antigens of cancer, including its antigenic portions, such as epitopes, that can optionally be expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)-binding peptide, such as 0.3 μM MART-1:26-35 (27L) or gpl00:209-217 (210M), in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens can include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TIL can also be rapidly expanded by restimulation with the same antigen of cancer pulsed on HLA-A2-expressing antigen-presenting cells. Alternatively, TIL can be further restimulated, for example, by irradiated autologous lymphocytes or by irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0214]

[0194] In one embodiment, the cell culture medium further contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU / mL of IL-2. In one embodiment, 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. In one embodiment, the cell culture medium contains IL-2 at a concentration of 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.

[0215]

[0195] In one embodiment, the cell culture medium contains the OKT3 antibody. In a preferred embodiment, the cell culture medium contains about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL and about 1 μg / mL of the OKT3 antibody. In one embodiment, the cell culture medium contains the OKT3 antibody at a concentration of 0.1 ng / mL - 1 ng / mL, 1 ng / mL - 5 ng / mL, 5 ng / mL - 10 ng / mL, 10 ng / mL - 20 ng / mL, 20 ng / mL - 30 ng / mL, 30 ng / mL - 40 ng / mL, 40 ng / mL - 50 ng / mL and 50 ng / mL - 100 ng / mL.

[0216]

[0196] In some embodiments, IL-2, IL-7, IL-15, and IL-21, and combinations thereof, can be included during the second expansion culture in the Step D process, as described herein.

[0217]

[0197] In some embodiments, the second expansion culture can be performed in an added cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells.

[0218]

[0198] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TIL to PBMC and / or antigen-presenting cells in the rapid expansion culture and / or the second expansion culture is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TIL to PBMC in the rapid expansion culture and / or the second expansion culture is 1:50 to 1:300. In one embodiment, the ratio of TIL to PBMC in the rapid expansion culture and / or the second expansion culture is 1:100 to 1:200.

[0219]

[0199] In one embodiment, the REP and / or the second expansion culture is performed in a flask by mixing bulk TIL with 100-fold or 200-fold excess inactivated feeder cells in 150 ml of medium, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2. Medium replenishment is performed until the cells are transferred to another growth chamber (generally 2 / 3 medium replenishment via respiration with fresh medium). Another growth chamber includes a GRex flask and a gas-permeable container, as further discussed in more detail below.

[0220]

[0200] In some embodiments, the second expansion culture (also referred to as the REP process) is shortened to 7 to 14 days, as discussed in the examples and figures. In some embodiments, the second expansion culture is shortened to 11 days.

[0221]

[0201] In one embodiment, REP and / or the second expansion culture can be carried out using a T-175 flask and a gas-permeable bag or a gas-permeable culture device (G-Rex flask) as described above (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42). In the rapid expansion culture of TIL and / or the second expansion culture in a T-175 flask, 1×10 6 TIL cells can be added to each T-175 flask suspended in 150 mL of medium. TIL can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU of IL-2 per mL and 30 ng of anti-CD3 per mL. The T-175 flask can be incubated at 37 °C in 5% CO2. Half of the medium can be replaced on day 5 with a 50 / 50 medium containing 3000 IU of IL-2 per mL. On day 7, the cells from two T-175 flasks are mixed in a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU of IL-2 per mL are added to 300 mL of the TIL suspension. The cell count in each bag is counted daily or every other day, and fresh medium is added to maintain the cell count at 0.5-2.0×10 6 cells / mL.

[0222]

[0202] In one embodiment, REP and / or the second expansion culture can be carried out in a 500 mL capacity gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) having a 100 cm 2 gas-permeable silicon bottom, and 5×10 6 or 10×10 6Individual TILs can be cultured with PBMCs in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU of IL-2 per mL, and 30 ng of anti-CD3 (OKT-3) per mL. G-Rex 100 flasks can be incubated at 37 °C under 5% CO2. On day 5, 250 mL of the supernatant can be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU of IL-2 per mL and returned to and added to the original G-Rex 100 flask. When continuously expanding TILs in G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 can be suspended in the 300 mL of medium present in each flask and the cell suspension can be divided into three 100 mL aliquots which can be used to seed three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU of IL-2 per mL can be added to each flask. The G-Rex 100 flasks can be incubated at 37 °C under 5% CO2 and 4 days later, 150 mL of AIM-V containing 3000 IU of IL-2 per mL can be added to each G-Rex 100 flask. The cells can be harvested on day 14 of culture.

[0223]

[0203] In one embodiment, the REP and / or the second expansion culture is performed in a flask by mixing bulk TILs with 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. Medium replenishment is performed until the cells are transferred to another growth chamber (generally 2 / 3 medium replenishment via respiration with fresh medium). The other growth chamber includes GRex flasks and gas permeable containers as will be considered further below.

[0224]

[0204] In one embodiment, REP and / or a second expansion culture is performed, which further includes the step of selecting TILs for excellent tumor responsiveness. Any selection method known in the art can be used. For example, the method described in US Patent Application Publication No. 2016 / 0010058A1 (the disclosure of which is incorporated herein by reference) can be used for the selection of TILs for excellent tumor responsiveness.

[0225]

[0205] The REP and / or the second expansion culture of TILs can be performed using a T-175 flask and a gas-permeable bag (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 a gas-permeable G-Rex flask as described above. In some embodiments, the REP and / or the second expansion culture is performed using a flask. In some embodiments, the REP is performed using a gas-permeable G-Rex flask. For TIL REP and / or the second expansion culture in a T-175 flask, about 1×10 6 TILs are suspended in about 150 mL of medium and added to each T-175 flask. The TILs are cultured at a ratio of 1 to 100 with irradiated allogeneic PBMCs as "feeder" cells, and the cells are cultured in a 1:1 mixture (50 / 50 medium) of CM and AIM-V medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. The T-175 flasks are incubated at 37 °C under 5% CO2. In some embodiments, half of the medium is replaced on day 5 with 50 / 50 medium containing 3000 IU / mL of IL-2. In some embodiments, on day 7, the cells from two T-175 flasks are combined into a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 is added to the 300 mL TIL suspension. The cell number of each bag can be counted daily or every two days, and fresh medium is added to keep the cell number at about 0.5 to about 2.0×106 can be maintained at cells / mL.

[0226]

[0206] 100 cm 2 For TIL REP and / or second expansion culture in a 500 mL capacity flask with gas-permeable silicon bottom (G-Rex 100, Wilson Wolf), in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3, about 5×10 6 or 10×10 6 individual TILs are cultured with irradiated allogeneic PBMCs at a ratio of 1 to 100. The G-Rex 100 flasks are incubated at 37 °C under 5% CO2. In some embodiments, on day 5, 250 mL of the supernatant is removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 g) for 10 minutes. Next, the TIL pellet can be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL of IL-2 and returned to and added to the original G-Rex 100 flask. In embodiments where the TILs are continuously expanded in the G-Rex 100 flask, on day 7, the TILs in each G-Rex 100 are suspended in the 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots, which are used to seed three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL of IL-2 is added to each flask. The G-Rex 100 flasks are incubated at 37 °C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 is added to each G-Rex 100 flask. The cells are harvested on day 14 of culture.

[0227] 1. Feeder cells and antigen-presenting cells

[0207] In one embodiment, the second expansion culture procedure (including steps D, REP) described herein requires an excess amount of feeder cells during REP TIL expansion culture and / or during the second expansion culture. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy donor. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

[0228]

[0208] Generally, allogeneic PBMCs are inactivated either by irradiation or heat treatment and are used in the REP procedure as described in the examples, particularly Example 14 which provides an exemplary protocol for evaluating the replicative ability of allogeneic PBMCs.

[0229]

[0209] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells transferred to the culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture), the PBMCs are considered non-replicating and are approved for use in the TIL expansion culture procedure described herein.

[0230]

[0210] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 has not increased from the initial number of viable cells transferred to the culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture) on days 7 and 14, the PBMCs are considered non-replicating and are approved for use in the TIL expansion culture procedure described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / ml of OKT3 antibody and 3000 IU / ml of IL-2.

[0231]

[0211] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 has not increased from the initial number of viable cells seeded on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start date of the second expansion culture) on days 7 and 14, the PBMCs are considered non-replicating and are approved for use in the TIL expansion culture procedures described herein. In some embodiments, PBMCs are cultured in the presence of 5 - 60 ng / ml of OKT3 antibody and 1000 - 6000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 10 - 50 ng / ml of OKT3 antibody and 2000 - 5000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 20 - 40 ng / ml of OKT3 antibody and 2000 - 4000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 25 - 35 ng / ml of OKT3 antibody and 2500 - 3500 IU / ml of IL-2.

[0232]

[0212] In one embodiment, the artificial antigen presenting cells are used at the REP stage as an alternative to or in combination with PBMCs.

[0233] 2. Cytokine

[0213] The expansion culture methods described herein generally use a culture medium containing high doses of cytokines, particularly IL-2, as is known in the art.

[0234] Instead, it is further possible to use a cytokine combination for the rapid expansion culture of TILs or the second expansion culture, and for combinations of two or more of IL-2, IL-15, and IL-21, generally as outlined in US Patent Application Publication No. 2017 / 0107490A1, International Publication No. 2015 / 189356, US Patent Application Publication No. 2017 / 0107490A1, and International Publication No. 2015 / 189357 (each of which is expressly incorporated herein by reference in its entirety). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latter finding particular use in many embodiments. As described in these specifications, using a cytokine combination is particularly advantageous for the production of lymphocytes, specifically T cells.

[0235] 3. Anti-CD3 antibody

[0215] In some embodiments, the culture medium (including REP) used in the expansion culture method described herein also contains an anti-CD3 antibody. When an anti-CD3 antibody is used in combination with IL-2, T cell activation and cell division are induced in the TIL population. This effect can be seen with full-length antibodies as well as Fab and F(ab’)2 fragments, with the former generally being preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719 (which is incorporated herein by reference in its entirety).

[0236]

[0216] As will be understood by those skilled in the art, suitable anti-human CD3 antibodies found to be useful in the present invention include, but are not limited to, anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including 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).

[0237] E. Step E: Recovery of TILs

[0217] After the second expansion culture step, the cells can be recovered. In some embodiments, the TILs are recovered after 1, 2, 3, 4 or more than 4 second expansion culture steps.

[0238]

[0218] The TILs can be recovered by any suitable and aseptic method, including for example by centrifugation. TIL recovery methods are well known in the art and any such known method can be used with the present process. In some embodiments, the TILs are recovered using an automated system. In some embodiments, the TILs are recovered using a semi-automated system. In some embodiments, the TILs are recovered using a semi-automated system. In some embodiments, the TILs from the second expansion culture are recovered using a semi-automated device. In some embodiments, the LOVO system is used (e.g., commercially available from Benchmark Electronics). In some embodiments, the recovery step includes washing the TILs, formulating the TILs and / or aliquoting the TILs. In some embodiments, the cells are optionally frozen after recovery or as part of the recovery.

[0239] F. Step F: Final formulation and / or transfer to an infusion bag

[0219] After steps A - E are completed, the cells are transferred to a container for use in administration to a patient.

[0240]

[0220] In one embodiment, the TILs expanded using the APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. The TILs expanded using the PBMCs of the present disclosure can be administered by any suitable route as known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, which is preferably continued for about 30 - 60 minutes. Other suitable administration routes include intraperitoneal, intrathecal and intralymphatic.

[0241] G. Additional expansion culture step As will be understood, any of the above steps A to F can be repeated any number of times and can be carried out in an order different from the above.

[0242]

[0222] In some embodiments, one or more expansion culture steps may be repeated before the final formulation step F. Such additional expansion culture steps may include elements of the above-described first and / or second expansion culture steps (e.g., including the components described in the cell culture medium). The additional expansion culture steps may further include additional elements including additional components in the cell culture medium that are supplemented to the cell culture medium before and / or during the additional expansion culture step.

[0243]

[0223] In a further embodiment, a cryopreservation step may precede or follow any of the expansion culture steps described in FIG. 23 and the above paragraphs, in which the cells generated during the expansion culture step are preserved using methods known in the art until they are required for the remaining steps of the manufacturing / expansion culture process.

[0244] Pharmaceutical compositions, dosages and dosing regimens of TIL, MIL and PBL

[0240] In one embodiment, TIL expanded using the methods of the present disclosure is administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TIL expanded using the methods of the present disclosure can be administered by any suitable route known in the art. Preferably, the TIL is administered as a single intra-arterial or intravenous infusion, which preferably lasts for approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal administration, intrathecal administration, and intralymphatic administration.

[0245]

[0241] Any suitable dose of TIL can be administered. Particularly when the cancer is a hematological malignancy, preferably an average of about 7.8×10 10 TIL of about 2.3×10 10 ~ about 13.7×10 10 TIL is administered. In one embodiment, about 1.2×10 10~about 4.3×10 10 of TILs are administered.

[0246]

[0242] 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 , 5×1011 , 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 are. In one embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 1×10 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 are in the range of. In one embodiment of the present invention, the number of TILs provided in the pharmaceutical composition of the present invention is about 4×10 8 ~about 2.5×10 9is within the range. In another embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 9.5×10 8 In another embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 4.1×10 8 In another embodiment, the number of TILs provided in the pharmaceutical composition of the present invention is 2.2×10 9 .

[0247]

[0243] In one embodiment of the present invention, the number of TILs provided by the pharmaceutical composition of the present invention is about 0.1×10 9 to about 15×10 9 TILs, about 0.1×10 9 to about 15×10 9 TILs, about 0.12×10 9 to about 12×10 9 TILs, about 0.15×10 9 to about 11×10 9 TILs, about 0.2×10 9 to about 10×10 9 TILs, about 0.3×10 9 to about 9×10 9 TILs, about 0.4×10 9 to about 8×10 9 TILs, about 0.5×10 9 to about 7×10 9 TILs, about 0.6×10 9 to about 6×10 9 TILs, about 0.7×10 9 to about 5×10 9 TILs, about 0.8×10 9 to about 4×10 9 TILs, about 0.9×10 9 to about 3×10 9 TILs or about 1×10 9 to about 2×10 9 TILs.

[0248]

[0244] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is, for example, lower than 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%, 0.04%, 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 0.0001% w / w, w / v or v / v of the pharmaceutical composition.

[0249]

[0245] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is higher than 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%, 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 0.0001% w / w, w / v or v / v of the pharmaceutical composition.

[0250]

[0246] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is within the range of 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 0.09% 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 of the pharmaceutical composition.

[0251]

[0247] In some embodiments, the concentration of TILs provided in the pharmaceutical composition of the present invention is within 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%, about 0.1% to about 0.9% w / w, w / v or v / v of the pharmaceutical composition.

[0252] In some embodiments, the amount of TILs provided in the pharmaceutical composition of the present invention is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g or less.

[0253]

[0249] In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 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 10 g.

[0254]

[0250] The TIL provided in the pharmaceutical composition of the present invention is effective over a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the weight of the subject to be treated, as well as the preference and experience of the attending physician. Clinically established dosages of TIL may also be used when appropriate. The amount of the pharmaceutical composition administered using the methods herein, such as the dosage of TIL, will depend on the human or mammal being treated, the severity of the disease or condition, the rate of administration, the nature of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

[0255]

[0251] In some embodiments, TIL can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, TIL can be administered in multiple doses. The administration can be once, twice, three times, four times, five times, six times or more than six times a year. The administration can be once a month, once every two weeks, once a week or every other day. The administration of TIL can be continued as long as necessary.

[0256]

[0252] In some embodiments, the effective dosage of TIL 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 , 6×1010 , 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 are. In some embodiments, the effective dosage of TIL is 1×10 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 ~5×1012 and 5×10 12 ~1×10 13 are in the range of.

[0257]

[0253] In one embodiment of the present invention, the clinical dose of MIL useful for a patient having acute myeloid leukemia (AML) is from about 4×10 8 to about 2.5×10 9 within the range of MIL. In another embodiment, the number of MIL provided in the pharmaceutical composition of the present invention is 9.5×10 8 MIL. In another embodiment, the number of MIL provided in the pharmaceutical composition of the present invention is 4.1×10 8 . In another embodiment, the number of MIL provided in the pharmaceutical composition of the present invention is 2.2×10 9 .

[0258]

[0254] In some embodiments, the effective dose of TIL is from about 0.01 mg / kg to about 4.3 mg / kg, from about 0.15 mg / kg to about 3.6 mg / kg, from about 0.3 mg / kg to about 3.2 mg / kg, from about 0.35 mg / kg to about 2.85 mg / kg, from about 0.15 mg / kg to about 2.85 mg / kg, from about 0.3 mg to about 2.15 mg / kg, from about 0.45 mg / kg to about 1.7 mg / kg, from about 0.15 mg / kg to about 1.3 mg / kg, from about 0.3 mg / kg to about 1.15 mg / kg, from about 0.45 mg / kg to about 1 mg / kg, from about 0.55 mg / kg to about 0.85 mg / kg, from about 0.65 mg / kg to about 0.8 mg / kg, from about 0.7 mg / kg to about 0.75 mg / kg, from about 0.7 mg / kg to about 2.15 mg / kg, from about 0.85 mg / kg to about 2 mg / kg, from about 1 mg / kg to about 1.85 mg / kg, from about 1.15 mg / kg to about 1.7 mg / kg, from about 1.3 mg / kgmg to about 1.6 mg / kg, from about 1.35 mg / kg to about 1.5 mg / kg, from about 2.15 mg / kg to about 3.6 mg / kg, from about 2.3 mg / kg to about 3.4 mg / kg, from about 2.4 mg / kg to about 3.3 mg / kg, from about 2.6 mg / kg to about 3.15 mg / kg, from about 2.7 mg / kg to about 3 mg / kg, from about 2.8 mg / kg to about 3 mg / kg or from about 2.85 mg / kg to about 2.95 mg / kg.

[0259]

[0255] In some embodiments, the effective dosage of TIL is in the range of 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 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg or about 198 to about 207 mg.

[0260]

[0256] The effective amount of TIL can be administered either as a single dose or multiple doses by any of the acceptable modes of administration of drugs having similar utility, including intranasal and transdermal routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, injection directly into the transplant or tumor, or inhalation.

[0261] Method for treating cancer

[0257] The compositions and combinations of the above-mentioned TIL, PBL and / or MIL (and their populations) can be used in methods for treating hyperproliferative disorders. In a preferred embodiment, they are for use in the treatment of cancer. In a preferred embodiment, the present invention provides a method for treating cancer, wherein the cancer is a hematological malignancy such as a liquid tumor. In a preferred embodiment, the present invention provides a method for treating cancer, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, relapsed and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0262]

[0258] In one embodiment, the present invention provides a method for treating cancer, wherein the cancer is a hematological malignancy responsive to treatment with a PD-1 and / or PD-L1 inhibitor comprising pembrolizumab, nivolumab, durvalumab, avelumab or atezolizumab.

[0263]

[0259] In one embodiment, the present invention provides a method for treating cancer using a population of tumor-infiltrating lymphocytes (TIL), (a) obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor comprises a first population of TIL; (b) optionally fragmenting or separating the tumor to obtain tumor fragments and contacting the tumor fragments with a first cell culture medium; (c) A step of performing initial expansion culture of a first population of tumor infiltrating lymphocytes (TIL) in a first cell culture medium to obtain a second population of TIL, wherein the second population of TIL is at least five times more numerous than the first population of TIL in number, and the first cell culture medium contains IL-2; (d) A step of performing second expansion culture of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the third population of TIL is at least 50 times more numerous than the second population of TIL in number 7 days after the start of the second expansion culture, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the second expansion culture is performed over a period of 14 days or less; (e) A step of recovering the third population of TIL; and (f) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, wherein the tumor is a liquid tumor and the cancer is a hematological malignancy, a method is provided.

[0264]

[0260] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), (a) A step of obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor contains a first population of TIL; (b) Optionally, fragmenting or separating the tumor to obtain tumor fragments and contacting the tumor fragments with a first cell culture medium; (c) A step of performing initial expansion culture of a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the second population of TIL is at least five times more numerous than the first population of TIL in number, and the first cell culture medium contains IL-2; (d) A step of performing a second expansion culture of a second population of tumor-infiltrating lymphocytes (TIL) in a second cell culture medium to obtain a third population of TIL, wherein the third population of TIL is at least 50 times more numerous than the second population of TIL 7 days after the start of the second expansion culture, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the second expansion culture is performed over a period of 14 days or less; (e) A step of collecting the third population of TIL; and (f) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer comprising, the tumor is a liquid tumor, and the cancer is selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, relapsed and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma, and provides a method which is a hematological malignancy.

[0265]

[0261] In one embodiment, the present invention is a method of treating cancer using a population of tumor-infiltrating lymphocytes (TIL), (a) A step of pre-treating a patient with a regimen comprising a kinase inhibitor or an ITK inhibitor; (b) A step of obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor contains a first population of TIL; (c) Optionally, fragmenting or separating the tumor to obtain tumor fragments and contacting the tumor fragments with a first cell culture medium; (d) A step of performing initial expansion culture of a first population of tumor infiltrating lymphocytes (TIL) in a first cell culture medium to obtain a second population of TIL, wherein the second population of TIL is at least five times more numerous than the first population of TIL in number, and the first cell culture medium contains IL-2; (e) A step of performing second expansion culture of the second population of TIL in a second cell culture medium to obtain a third population of TIL, wherein the third population of TIL is at least 50 times more numerous than the second population of TIL in number 7 days after the start of the second expansion culture, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMC), and the second expansion culture is performed over a period of 14 days or less; (f) A step of recovering the third population of TIL; and (g) A step of administering a therapeutically effective portion of the third population of TIL to a patient having cancer Including, the tumor is a liquid tumor and the cancer is a hematological malignancy, a method is provided.

[0266]

[0262] In one embodiment, the present invention is a method for treating cancer using a population of tumor infiltrating lymphocytes (TIL), (a) A step of pre-treating a patient with a regimen containing a kinase inhibitor or an ITK inhibitor; (b) A step of obtaining a tumor from a patient by resection, biopsy, needle aspiration or apheresis, wherein the tumor contains a first population of TIL; (c) Optionally, fragmenting or separating the tumor to obtain tumor fragments and contacting the tumor fragments with a first cell culture medium; (d) A step of performing initial expansion culture of a first population of TIL in a first cell culture medium to obtain a second population of TIL, wherein the second population of TIL is at least five times more numerous than the first population of TIL in number, and the first cell culture medium contains IL-2; (e) A step of performing a second expansion culture of a second population of TILs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50 times more numerous than the second population of TILs 7 days after the start of the second expansion culture, the second cell culture medium contains IL-2, OKT-3 (anti-CD3 antibody) and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), and the second expansion culture is performed over a period of 14 days or less; (f) A step of collecting the third population of TILs; and (g) A step of administering a therapeutically effective portion of the third population of TILs to a patient having cancer comprising, the tumor is a liquid tumor, and the cancer is selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, recurrent and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myelogenous leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma, a method is provided.

[0267]

[0263] In one embodiment of the present invention, TILs are expanded using the MIL method 1 and administered to a patient according to the present invention.

[0268]

[0264] In one embodiment of the present invention, TILs are expanded using the MIL method 2 and administered to a patient according to the present invention for treating cancer.

[0269]

[0265] In one embodiment of the present invention, TIL is expanded using the MIL method 3 and administered to a patient according to the present invention for treating cancer.

[0270]

[0266] In one embodiment of the present invention, TIL expanded using the MIL method 1, the MIL method 2, or the MIL method 3 is administered to a patient according to the present invention for treating AML.

[0271]

[0267] In one embodiment of the present invention, TIL is expanded using the PBL method 2 and administered to a patient according to the present invention for treating cancer.

[0272]

[0268] In one embodiment of the present invention, TIL is expanded using the PBL method 2 and administered to a patient according to the present invention for treating cancer.

[0273]

[0269] In one embodiment of the present invention, TIL is expanded using the PBL method 2 and administered to a patient according to the present invention for treating cancer.

[0274]

[0270] In one embodiment of the present invention, TIL expanded using the PBL method 1, the PBL method 2, or the PBL method 3 is administered to a patient according to the present invention for treating CLL.

[0275]

[0271] In any of the foregoing embodiments of the present invention, pretreatment with a kinase inhibitor is described. In one embodiment, the kinase inhibitor is selected from the group consisting of imatinib, dasatinib, ibrutinib, bosutinib, nilotinib, erlotinib, or other kinase inhibitors, tyrosine kinase inhibitors, or serine / threonine kinase inhibitors known in the art. In one embodiment, the pretreatment regimen with the kinase inhibitor is as known in the art and / or as directed by a physician.

[0276]

[0272] In any of the foregoing embodiments of the present invention, pretreatment with an interleukin-2-inducible T-cell kinase inhibitor (ITK) is described. Interleukin-2-inducible T-cell kinase (ITK) is a non-receptor tyrosine kinase expressed in T cells and regulates various pathways. Any ITK inhibitor known in the art can be used in the embodiments of the present invention (for example, Lo, et al., Expert Opinion on Therapeutic Patents, 20:459-469 (2010); Vargas, et al, Scandinavian Journal of Immunology, 78(2):130-139 (2013); International Publication No. 2015112847; International Publication No. 2016118951; International Publication No. 2007136790, US Patent Application Publication No. 20120058984A1 and US Patents Nos. 9,531,689 and 9,695,200; all of which are hereby incorporated by reference in their entirety). In one embodiment of the present invention, the ITK inhibitor is a covalent ITK inhibitor that binds covalently and irreversibly to ITK. In one embodiment of the present invention, the ITK inhibitor is an allosteric ITK inhibitor that binds to ITK.In one embodiment of the present invention, the ITK inhibitor is selected from the group consisting of aminothiazole-based ITK inhibitors, 5-aminomethylbenzimidazole-based ITK inhibitors, 3-aminopyrid-2-one-based ITK inhibitors, (4- or 5-aryl)pyrazolyl-indole-based ITK inhibitors, benzimidazole-based ITK inhibitors, aminobenzimidazole-based ITK inhibitors, aminopyrimidine-based ITK inhibitors, aminopyridine-based ITK inhibitors, diazolodiazine-based ITK inhibitors, triazole-based ITK inhibitors, 3-aminopyrid-2-one-based ITK inhibitors, indolylindazole-based ITK inhibitors, indole-based ITK inhibitors, azaindole-based ITK inhibitors, pyrazolylindole-based inhibitors, thienopyrazole-based ITK inhibitors, heterocyclic ITK inhibitors, and ITK inhibitors targeting cysteine-442 in the ATP pocket (such as ibrutinib), azabenzimidazole-based ITK inhibitors, benzothiazole-based ITK inhibitors, indole-based ITK inhibitors, pyridone-based ITK inhibitors, sulfoximine-substituted pyrimidine ITK inhibitors, arylpyridinone-based ITK inhibitors, and any other ITK inhibitor known in the art. In one embodiment of the present invention, the pretreatment regimen with the ITK inhibitor is as known in the art and / or as directed by a physician. In one embodiment of the present invention, the ITK inhibitor is...

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[0277]

[0273] In any of the foregoing embodiments, a pretreatment regimen comprising ibrutinib (commercially available as IMBRUVICA, chemical name 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one) includes orally administering 1 capsule of 140 mg 4 times a day (q.d.) for a period of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months or 6 months; orally administering 2 capsules of 140 mg 4 times a day; orally administering 3 capsules of 140 mg 4 times a day; or orally administering 4 capsules of 140 mg 4 times a day. In the foregoing embodiments, a pretreatment regimen comprising ibrutinib may also include orally administering an ibrutinib dosage selected from the group consisting of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg and 500 mg, wherein the administration is carried out 1 time a day, 2 times a day, 3 times a day or 4 times a day, and the administration period is selected from the group consisting of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months and 6 months.

[0278]

[0274] In any of the foregoing embodiments, the cancer to be treated is a hematological malignancy selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, relapsed and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

[0279]

[0275] The effectiveness of the methods and compositions described herein in the treatment, prevention, and / or management of the indicated diseases or disorders can be tested using various animal models known in the art.

[0280] Myeloablative lymphodepletion by chemotherapy

[0276] In one embodiment, the present invention provides a method of treating cancer with a TIL population, wherein the patient is pre-treated with myeloablative chemotherapy prior to infusion of the TILs according to the present disclosure. In one embodiment, the myeloablative chemotherapy is one or more chemotherapeutic agents. In one embodiment, the myeloablative chemotherapy is cyclophosphamide at 60 mg / kg / day for 2 days (27 and 26 days prior to TIL infusion) and fludarabine at 25 mg / m 2 / day for 5 days (27 - 23 days prior to TIL infusion). In one embodiment, after myeloablative chemotherapy and TIL infusion (day 0) according to the present disclosure, the patient receives intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours up to physiological tolerance.

[0281]

[0277] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (the "cytokine sink"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as an "immunosuppression adjustment") in a patient prior to introduction of the TILs of the present invention.

[0282]

[0278] Generally, lymphodepletion is achieved using administration of fludarabine or cyclophosphamide (the active form is called mafosfamide) and combinations thereof. Such methods are described in Gassner, et al, Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al, Nat. Clin. Pract. Oncol, 2006, 3, 668-681, Dudley, et al, J. Clin. Oncol. 2008, 26, 5233-5239 and Dudley, et al, J. Clin. Oncol. 2005, 23, 2346-2357, all of which are hereby incorporated by reference in their entirety.

[0283]

[0279] In some embodiments, fludarabine is administered at a concentration of 0.5 μg / mL to 10 μg / mL fludarabine. In some embodiments, fludarabine is administered at a concentration of 1 μg / mL fludarabine. In some embodiments, fludarabine treatment is performed 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, fludarabine treatment is performed at 35 mg / kg / day for 2 to 7 days. In some embodiments, fludarabine treatment is performed at 35 mg / kg / day for 4 to 5 days. In some embodiments, fludarabine treatment is performed at 25 mg / kg / day for 4 to 5 days.

[0284]

[0280] In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 0.5 μg / mL to 10 μg / mL by administration of cyclophosphamide. In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 1 μg / mL by administration of cyclophosphamide. In some embodiments, the cyclophosphamide treatment is carried out for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days or more. In some embodiments, cyclophosphamide is 2 administered at a dose of 100 mg / m 2 / day, 150 mg / m 2 / day, 175 mg / m 2 / day, 200 mg / m 2 / day, 225 mg / m 2 / day, 250 mg / m 2 / day, 275 mg / m 2 / day or 300 mg / m 2 / day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.v.). In some embodiments, the cyclophosphamide treatment is carried out at 35 mg / kg / day for 2 to 7 days. In some embodiments, the cyclophosphamide treatment is carried out at 250 mg / m 2 / day, i.v. for 4 to 5 days. In some embodiments, the cyclophosphamide treatment is carried out at 250 mg / m 2 / day, i.v. for 4 days.

[0285]

[0281] In some embodiments, lymphodepletion is performed by administering both fludarabine and cyclophosphamide to the patient. In some embodiments, over 4 days, fludarabine is administered at 25 mg / m 2 / day, i.v., and cyclophosphamide is administered at 250 mg / m 2 / day, i.v.

[0286]

[0224] In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg / m 2 / day for 2 days, followed by 25 mg / m2 It is carried out by administering fludarabine at a dose of 60 mg / m 2 per day for 5 days. Some methods for expanding and culturing TIL obtained from bone marrow or peripheral blood are described herein. In one embodiment of the present invention, lymphodepletion is carried out by administering cyclophosphamide at a dose of 25 mg / m 2 per day for 2 days, followed by administering fludarabine at a dose of 25 mg / m

Examples

[0287] Examples

[0224] The embodiments included in this specification are described with reference to the following examples. These examples are provided for illustrative purposes only, and the disclosure included in this specification should in no way be construed as being limited to these examples, but rather should be construed as encompassing all possible variations that become apparent as a result of the teachings provided herein.

[0288] Example 1 - Expansion and Culture of TIL from Non-Hodgkin Lymphoma

[0282] TIL was expanded and cultured from 5 non-Hodgkin lymphoma tumors (1 mantle cell lymphoma tumor, 3 follicular lymphoma tumors, and 1 ABC-type diffuse large B-cell lymphoma tumor) having the pathological conditions shown in FIG. 1 using IL-2 for 11-14 days at the pre-REP stage, followed by performing REP for 14 days using IL-2, mitogen anti-CD3 antibody, and irradiated allogeneic peripheral blood mononuclear cell (PBMC) feeders. TIL was successfully generated from all 5 lymphoma tumors, and the maximum expansion culture index was 680-fold, which was significantly higher than that previously observed using other methods. Schwartzentruber, et al., Blood 1993, 82, 1204-1211. Furthermore, the average CD3 + T cell population was 95% (compared to 75% using the method of Schwartzentruber, et al., Blood 1993, 82, 1204-1211).

[0289]

[0283] Cell sorting and flow cytometry were performed using a Becton, Dickinson & Co. (BD) FACS CANTO II system. By flow cytometry analysis, a marked relative increase in effector memory cells comparable to those of melanoma TILs was observed (Figure 2). Compared to melanoma TIL cultures, a significant increase in effector memory CD45RA+ (TEMRA) cells (p = 0.0013; CD4, CD8) and CD28+CD4+ (p = 0.008) subsets was observed in lymphoma (Figure 3).

[0290]

[0284] CD4 + and CD8 + Comparisons of phenotypic markers of T cell differentiation in the CD4 + and CD8 + subsets are shown in Figures 4 and 5, respectively. Comparisons of phenotypic markers of T cell exhaustion in the CD4

[0291]

[0285] Figure 8 shows a comparison of cell types between non-Hodgkin lymphoma TILs and melanoma TILs. A tendency for an increase in the number of CD4 + T cells in lymphoma TILs compared to melanoma TILs is shown.

[0292]

[0286] Figure 9 shows the results of a bioluminescence redirected lysis assay (BRLA). Compared to melanoma TILs (11 - 75 LU 50 , 4 hours), in lymphoma TILs, the minimal cell lysis activity of TILs measured by BRLA as LU 50 / 10 6 was in the range of less than 1 to 6 LU 50 at 4 hours and 1 to 39 LU 50 at 24 hours.

[0293]

[0287] Figure 10 shows the results of an enzyme-linked immunosorbent assay (ELISA) for interferon-γ (IFN-γ) of lymphoma tumor-infiltrating lymphocytes (TILs) versus melanoma TILs. Comparable results are shown. The results of the ELISpot assay of lymphoma TILs are shown in Figure 11 and compared to the results of the same assay for melanoma TILs in Figure 12. In the ELISpot assay, extensive IFN-γ production by lymphoma TILs was observed upon stimulation with phorbol 12-myristate 13-acetate / ionomycin, anti-CD3 antibody, or CD3 / CD28 / 4-1BB beads, and the IFN-γ produced by some lymphoma TILs under these conditions was equivalent to that produced by melanoma TILs, and in some cases, IFN-γ production in lymphoma TILs was much higher.

[0294]

[0288] Figure 13 shows the results of NANOSTRING NCOUNTER analysis (Nanostring Technologies, Inc., Seattle, WA), which shows that lymphoma TILs express higher levels of RORC IL17A (TH17 phenotype) and GATA3 (Th2 phenotype) compared to melanoma TILs. This finding is consistent with the observation that lymphoma-reactive T cells are predominantly TH2 and TH17.

[0295]

[0289] Overall, the results provide evidence that TIL cell therapy can be used for the treatment of lymphoma patients.

[0296] Example 2 - Phenotypic and Functional Characterization of Myeloid Infiltrating Lymphocytes (MILs) Expanded from the Bone Marrow of AML Patients and Peripheral Blood Lymphocytes (PBLs) Expanded from the Peripheral Blood of AML Patients

[0290] Bone marrow samples and available related blood samples are obtained from patients with acute myeloid leukemia (AML) who have been pre-treated with at least 3 rounds of a regimen containing ibrutinib (1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one), along with information regarding the patient's age, gender, stage, tumor type, cancer site, treatment history, anonymized pathology reports, and molecular tests performed (e.g., MSI expression and Raf / Ras expression). MIL and PBL are expanded using one of MIL method 1, MIL method 2, or MIL method 3 or PBL method 1, PBL method 2, or PBL method 3, and the phenotypes and functions of MIL and PBL are characterized.

[0297]

[0291] Figures 36A and 36B show the magnification of MIL and PBL. Figure 36A shows the magnification of three patients (MIL1, MIL2, MIL3), and Figure 36B shows the magnification of the matched PBL of patients 2 and 3 (PBL2, PBL3). MIL1.1 was expanded using MIL method 1, MIL1.2 was expanded using MIL method 2, MIL1.3 was expanded using MIL method 3, and PBL was expanded using PBL method 3. The magnification of MIL1 shows an increase of 25 (MIL1.1), 50 (MIL1.2), and 75 (MIL1.3) times for each sample within MIL1. This preliminarily indicates that MIL method 3 may be the preferred expansion method. The magnification data for MIL2 and MIL3 appear to be insufficient. This may be due to a low number of starting cells. For comparison, the starting cell number of sample 3 of patient MIL1 (MIL1.3) was 138,000 cells, while the starting cell numbers of MIL2 and MIL3 were 62,000 and 28,000, respectively. The magnification of PBL shown in Figure 36B for MIL2 and MIL3 was approximately 10 times and 40 times, respectively, with similar starting cell numbers (338,000 for PBL2 and 336,000 for PBL3).

[0298] [

[0292] ] Figures 37A and 37B show the number of IFN-γ-producing cells in MIL (Figure 37A) and compatible PBL (Figure 37B). MIL1.3, MIL2, and MIL3 show a significant increase in IFN-γ secretion, indicating that MIL method 3 is the preferred expansion culture method. The PBL data are inconclusive.

[0299] [

[0293] ] Figures 38A - 38F show the TCRαβ+, CD4+, and CD8+ subsets of MIL and PBL. Figures 38A and 38D show the TCRab+ subset of MIL expanded using all three methods (MIL1.1, MIL1.2, MIL1.3) (Figure 38A) and PBL expanded using PBL method 3 (Figure 38D). The data show that the TCRαβ+ subset is approximately 100% in all MIL and PBL, indicating that the expansion culture process was successful in the expansion culture of almost all T cells. Figures 38B and 38E show a decrease in the CD4 subset for MIL expanded by MIL method 3 (which correlates with the increase in the CD8 subset in Figure 38C). The PBL data in Figures 38E and 38F appear to be consistent with the MIL1.3 data.

[0300]

[0301] ​Figures 41A - D and 42A - D show data for the CD8 subsets of MIL (Figure 41) and PBL (Figure 42). Figures 41A and 42A show data for naive (CCR7+ / CD45RA+); Figures 41B and 42B show data for central memory T cells (CM) (CCR7+ / CD45RA-); Figures 41C and 42C show data for effector memory T cells (EM) (CCR7- / CD45RA-); Figures 41D and 42D show data for terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+). Samples expanded using the MIL method 3 (MIL1.3) are consistent with the CD4 subset of melanoma TIL, which is the comparator. Data for PBL2 and PBL3 were used as controls.

[0302] Figures 43A and 43B show data for the CD4CD27 and CD8CD27 subsets of MIL (Figure 43A) and PBL (Figure 43B). Figures 44A and 44B show data for the CD4CD28 and CD8CD28 subsets of MIL (Figure 44A) and PBL (Figure 44B). PBL data are shown at day 0 and day 14 of the expansion culture process for each sample compared to melanoma TIL. MIL data are shown at day 0 and day 14 only for MIL1.3 compared to melanoma TIL. The CD28 subsets of MIL and PBL are similar to melanoma TIL.

[0303]

[0304] Figures 45A and 45B represent a comparison of PD1+ cells within each of the CD4 and CD8 subsets of MIL (Figure 45A) and PBL (Figure 45B). Figures 46A and 46B represent a comparison of LAG3+ cells within each of the CD4 and CD8 subsets of MIL (Figure 46A) and PBL (Figure 46B). Data for both PD1+ and LAG3+ show a significant decrease in the MIL1.3 sample relative to the day 0 measurement, while for MIL1.1 and MIL1.2, both PD1 and LAG3 tended to increase relative to day 0. PBL data were used as controls.

[0304]

[0298] The experiments of this example showed that the MIL expanded by the MIL method 3 had a higher expansion multiple, high functionality, a higher proportion of the CD8 subset, and fewer LAG3+ and PD1+ T cell subsets. The data also showed that the memory subset was similar to melanoma TIL. The data also showed that the cryopreserved samples seemed to have a higher expansion multiple compared to the fresh samples. Much of the data of the PBL samples seems to be based on a small sample size and is uncertain.

[0305] Example 3 - Method for expanding TIL and treating cancer using the expanded TIL

[0299] Bone marrow is obtained using needle aspiration. The bone marrow sample is aspirated into a heparin-containing syringe and stored overnight at room temperature. After storage, the contents of the syringe are pooled into a sterile container and tested for quality. The bone marrow is concentrated for mononuclear cells (MNC) using lymphocyte separation medium (LSM) and centrifugation by COBE Spectra. The cells within the gradient are collected up to the level of the erythrocyte pellet and washed using HBSS. The MNC are cryopreserved using a hetastarch-based cryoprotectant to which 2% HSA and 5% DMSO are added, and a portion of the MNC is reserved for quality control. The QC vial is thawed to measure the cell content of CD3 + and CD38 + / 138 + of the MNC product.

[0306]

[0300] The bone marrow is aspirated and fractionated on a lymphocyte separation medium density gradient, and the cells are collected to approximately the level of the erythrocyte pellet. This fractionation method substantially removes erythrocytes and neutrophils and provides a nearly complete bone marrow. The resulting fractionated material consists of T cells and tumor cells. The bone marrow is Ficolled, and the TIL are expanded using methods known in the art and any method described herein. For example, an exemplary method for expanding TIL is shown in FIG. 14. Exemplary methods for expanding TIL and treating cancer patients with the expanded TIL are shown in FIG. 15.

[0307] Example 4 - Phenotypic and Functional Characterization of Tumor-Infiltrating Lymphocytes (TILs) Proliferated from Non-Hodgkin Lymphoma Tumors

[0301] The goals of the experiments described in this example included determining whether therapeutically effective TILs could be isolated and cultured from NHL tumors and comparing the characteristics of NHL-derived TILs with those of melanoma-derived TILs.

[0308]

[0302] The materials and methods for the extraction and expansion culture of TILs from patients were as described herein. Patient TILs were extracted from the suppressive tumor microenvironment via surgical resection of the lesion, in this case lymphoid tissue. TILs were expanded using the expansion culture process disclosed herein to obtain 10 9 ~10 11 individual TILs.

[0309]

[0303] NHL-derived TILs (one mantle cell lymphoma (MCL), three follicular lymphomas (FL), three diffuse large B-cell lymphomas (DLBCL)) were analyzed for differentiation markers against melanoma-derived TILs using flow cytometry. TILs were analyzed for anti-CD56, anti-TCRab, anti-CD3, anti-CD4, anti-CD8, anti-CD27, and anti-CD28 antibodies. These antibodies were used as differentiation panel 1 (DF1). Anti-CD3, anti-CD4, anti-CD9, anti-CD38, anti-HLA-DR, anti-CCR7, and anti-CD45RA antibodies were used as differentiation panel 2 (DF2). DF2 was used to identify the following T cell subsets: naive (CCR7+ / CD45RA+); central memory T cells (CM) (CCR7+ / CD45RA-); effector memory T cells (EM) (CCR7- / CD45RA-); and terminally differentiated effector memory cells (TEMRA) (CCR7- / CD45RA+).

[0310]

[0304] Figure 16 shows CD4 and CD8 T cells in different cell subpopulations in different cancer types. Cancer types including melanoma (black), mantle cell (red), diffuse large B-cell lymphoma (blue), and follicular lymphoma (purple) were tested. Figures 16A - 16D generally show that lymphoma TILs tend to be more proliferative and thus have higher antitumor activity compared to melanoma TILs. Similarly, Figure 17B shows that CD4 / CD28-expressing lymphoma T cells have a higher proliferative capacity than CD4 / CD28-expressing melanoma T cells.

[0311]

[0305] Interferon gamma (IFNγ) production by TILs was measured by stimulating TILs with mAB-coated Dynabeads™ (CD3, CD28, and CD137) and using ELISpot™ (Immunospot CTL), enumerated by ELISA using an Immunospot™ S6 entry analyzer and also using a DuoSet™ ELISA kit (ELISA according to the manufacturer's instructions using R&D Systems).

[0312]

[0306] Figures 18A and 18B show that IFNγ production by NHL TILs and melanoma TILs is similar, indicating similar cytotoxic functions between the two TIL types.

[0313]

[0307] The lysis ability of TILs was determined using a bioluminescence redirected lysis assay (BRLA). P815 cells transduced with a lentiviral vector encoding eGFP and firefly luciferase were used as target cells. TILs and target cells were co-cultured for 4 hours / 24 hours in the presence of OKT3. Then, luciferin was added and the cells were incubated for 5 minutes. Bioluminescence was measured using a luminometer. Percent survival and percent cytotoxicity were calculated as follows. % survival = (experimental survival - minimum) / (maximum signal - minimum signal) × 100 % cytotoxicity = 100 - (% survival)

[0314]

[0308] The lytic ability of TILs is expressed as the lytic unit LU representing 50 percent cytotoxicity of target cells induced by effector cells. 50 It was expressed as.

[0315]

[0309] TILs were assayed to measure their tumor killing ability against both autologous tumors and allogeneic tumors. TILs were mixed with autologous lymphoma cells or an allogeneic melanoma cell line (526 melanoma cell line) at different effector cell to target cell ratios (E:T ratios) (either 10:1, 20:1, 50:1 or 100:1). Tumor cells were labeled with CellTrace Violet dye (ThermoFisher) before co-culture. After 24 hours, the cells were stained with 7-AAD to determine cell death. The percentage of tumor cells killed by TILs was represented as 7-AAD positive tumor cells gated by CellTrace Violet dye against CD19 for lymphoma cells and against MCSP for melanoma cells.

[0316]

[0310] Figure 19 shows that NHL TILs and melanoma TILs have similar cytotoxic functions against both allogeneic and autologous tumors at 4 hours (Figure 19A) and 24 hours (Figure 19B).

[0317]

[0311] Gene expression analysis was also performed on TILs using the nCounter GX Human Immunology V2 panel (NanoString, Seattle). 100 ng of total RNA was assayed according to the manufacturer's instructions. The data was normalized by scaling to the geometric mean of the endogenous control gene probes for each sample. The data was mapped and compared against melanoma gene expression.

[0318]

[0312] Figure 21 shows the results of the gene expression analysis. The heatmap shows the fold change in gene expression across melanoma TILs. The expression of IL17A and RORC from lymphoma-derived TILs showed higher expression compared to melanoma-derived TILs.

[0319]

[0313] Overall, the results of this experiment demonstrate that the functional characteristics of lymphoma-derived TILs are similar to those of melanoma-derived TILs, indicating that the use of lymphoma-derived TILs will likely succeed in treating lymphoma cancer.

[0320] Example 5 - Phenotypic and functional characterization of peripheral blood lymphocytes (PBLs) expanded from the peripheral blood of patients with chronic lymphocytic leukemia (CLL).

[0314] PBMCs were collected from patients with CLL before and after 3 rounds of treatment with ibrutinib.

[0321]

[0315] As described in FIG. 24 and elsewhere in this specification, T cells were expanded using three different methods, PBL method 1, PBL method 2, and PBL method 3. Certain samples were obtained from fresh PBMCs and certain samples were obtained from cryopreserved PBMCs. Once the cells were expanded and harvested, they were phenotyped and functionally characterized using the methods described in Example 4 and elsewhere in this specification. The goal of this example was to determine the optimal expansion process for PBLs and to determine whether PBLs expanded from ibrutinib-treated samples were more potent than PBLs expanded from untreated samples.

[0322]

[0316] The fold expansion of PBLs is shown in FIG. 26. Results for PBLs expanded using PBL method 1, PBL method 2, and PBL method 3 are shown. Untreated PBLs (pre-Rx PBLs) showed an average fold expansion of 179, and ibrutinib-treated PBLs (post-Rx PBLs) showed an average fold expansion of 306. PBLs derived from fresh PBMCs (PBL) showed an average fold expansion of only 82. p = 0.006 between PBL and post-Rx PBL. p = 0.3 between PBL and pre-Rx PBL and p = 0.1 between pre-Rx PBL and post-Rx PBL. Overall, an increase in average fold expansion was seen in all post-Rx PBL groups compared to all groups of both PBL and pre-Rx PBL.

[0323]

[0317] Figure 27 shows the interferon gamma (IFN-γ) - producing cells of PBL, pre - Rx PBL, and post - Rx PBL. For PBL, the average number of IFN - γ - producing cells was approximately 1864. For pre - Rx PBL, the average number of IFN - γ - producing cells was approximately 7530, and for post - Rx PBL, the average number of IFN - γ - producing cells was approximately 11984. The p - value between PBL and post - Rx PBL was 0.006. The p - value between PBL and pre - Rx PBL was 0.006 and the p - value between pre - Rx PBL and post - Rx PBL was 0.01. Overall, a significant increase in the average number of IFN - γ - producing cells was seen in all post - Rx PBL groups compared to all groups of both PBL and pre - Rx PBL.

[0324]

[0318] Phenotypic characterization was performed on each sample. Figure 28 shows the percentages of CD4+ and CD8+ T - cell subsets in pre - Rx PBL and post - Rx PBL, using melanoma TIL as a comparator. Here, the data show that the CD4 subset (shown on the left) is equivalent between pre - Rx PBL and post - Rx PBL regardless of the method used for cell expansion culture. The CD4 subsets of pre - Rx PBL and post - Rx PBL were shown to be higher than those of melanoma TIL (p = 0.0006 for each). The CD8 subset (shown on the right) was low in both pre - Rx PBL and post - Rx PBL regardless of the process used for cell expansion culture. The CD8 subsets of pre - Rx PBL and post - Rx PBL were shown to be lower than those of melanoma TIL (p = 0.0006 for each). The lower CD8 subset is hypothesized to be simply a derivative of the cancer type (i.e., in CLL, the CD4 subset is typically expanded).

[0325]

[0319] Figures 29A - 29D show the comparison between pre - Rx PBL and post - Rx PBL CD4 memory subsets using melanoma TIL as a comparator. Figure 29A shows data for naive (CCR7+ / CD45RA+); Figure 29B shows data for central memory T cells (CM) (CCR7+ / CD45RA - ); Figure 29C shows data for effector memory T cells (EM) (CCR7 - / CD45RA - ); Figure 29D shows data for terminally differentiated effector memory cells (TEMRA) (CCR7 - / CD45RA+). Figure 29 shows that the CD4 memory subsets of pre - Rx PBL and post - Rx PBL are equivalent to those seen in melanoma TIL.

[0326]

[0320] Figures 30A - 30D show the comparison between pre - Rx PBL and post - Rx PBL CD8 memory subsets using melanoma TIL as a comparator. Figure 30A shows data for naive (CCR7+ / CD45RA+); Figure 30B shows data for central memory T cells (CM) (CCR7+ / CD45RA - ); Figure 30C shows data for effector memory T cells (EM) (CCR7 - / CD45RA - ); Figure 30D shows data for terminally differentiated effector memory cells (TEMRA) (CCR7 - / CD45RA+). Figure 30 shows that the CD8 memory subsets of pre - Rx PBL and post - Rx PBL are equivalent to those seen in melanoma TIL.

[0327]

[0321] Figures 31A and 31B represent the comparison of CD27 subsets of CD4 cells (Figure 31A) and CD8 cells (Figure 31B) using melanoma TIL as a comparator. The CD4CD27 cell subset was significantly higher in both pre - Rx PBL (p = 0.03) and post - Rx PBL (p = 0.02) compared to melanoma TIL. The CD8CD27 cell subset was significantly higher in both pre - Rx PBL (p = 0.002) and post - Rx PBL (p = 0.001) compared to melanoma TIL.

[0328]

[0322] Figures 32A and 32B represent the comparison of the CD28 subsets of CD4 cells (Figure 30A) and CD8 cells (Figure 30B) using melanoma TIL as a comparator. The CD4CD28 cell subset and the CD8CD28 cell subset were shown to be equivalent in both pre-Rx PBL and post-Rx PBL compared to melanoma TIL.

[0329]

[0323] Figures 33A and 33B show the comparison of the LAG3+ subsets within the CD4+ (Figure 33A) and CD8+ (Figure 33B) populations of pre-Rx PBL and post-Rx PBL. The data show a significant mean decrease in the LAG3+ subset in both the CD4+ (p = 0.06) and CD8+ (p = 0.01) populations of post-Rx PBL.

[0330]

[0324] Figures 34A and 34B show the comparison of the PD1+ subsets within the CD4+ (Figure 34A) and CD8+ (Figure 34B) populations of pre-Rx PBL and post-Rx PBL. The data show a mean decrease in the PD1+ subset in both the CD4+ and CD8+ populations of post-Rx PBL, but the decrease was not significant.

[0331]

[0325] Figures 35A and 35B show the results of the cytolytic activity of pre-Rx PBL (Figure 35A) and post-Rx PBL (Figure 35B) measured using a bioluminescence re-dissolution assay (BRLA). The assay was performed as follows using the CelllTrace™ Violet Cell Proliferation Kit (Invitrogen). Effector cells, which were PBL, were labeled with carboxyfluorescein succinimidyl ester (CFSE). Target cells (autologous CD19+ tumor cells) were incubated with mitosiyin C and labeled with CellTrace Vioet Cell Proliferation Kit according to the instructions with CellTrace™ Violet (CTV). Effector cells and target cells were incubated for 24 hours at ratios of 2:1, 5:1, 20:1 (E:T cells). Countbright beads were added, the cells were stained with annexin V-PI, and CTV+ / annexin-V PI+ cells (providing the number of dead cells) were analyzed. Post-Rx PBL is thought to be more potent because fewer cells are required to kill 50% of the target tumor cells (i.e., LU 50 is lower for post-Rx PBL than for pre-Rx PBL).

[0332]

[0326] The experiments conducted in this example showed the following results: PBL expanded from freshly isolated CLL PBMC showed a lower expansion fold and significantly lower IFN-γ production compared to PBL expanded from cryopreserved PBMC (pre-Rx PBL and post-Rx PBL); post-Rx PBL consistently showed a higher expansion fold and a significant increase in IFN-γ production compared to pre-Rx PBL; both pre-Rx PBL and post-Rx PBL showed cytolytic activity against autologous (CD19+) tumor cells, but post-Rx PBL had a lower LU 50 than pre-Rx PBL.

[0333]

[0327] The examples shown above are provided to give those skilled in the art a complete disclosure and description of how embodiments of the compositions, systems, and methods of the present invention can be made and used, and are not intended to limit the scope of what the inventors regard as their invention. Variations of the above-described aspects for carrying out the present invention that will be apparent to those skilled in the art are intended to be within the scope of the following claims. All patents and publications referred to herein are indicative of the level of skill of those skilled in the art to which the present invention pertains.

[0334]

[0328] All headings and section designations are used for clarity only and are to be regarded as merely for reference and in no way limiting. For example, those skilled in the art will understand the utility of appropriately combining various aspects from different headings and sections in accordance with the spirit and scope of the invention described herein.

[0335]

[0329] All references cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0336]

[0330] As will be apparent to those skilled in the art, many modifications and variations of the present application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are provided by way of illustration only, and the present application should be limited only by the terms of the appended claims together with the full scope of equivalents recognized in the claims.

Claims

1. A method for expanding peripheral blood lymphocytes (PBL) from peripheral blood, comprising: a. obtaining a sample of peripheral blood mononuclear cells (PBMC) from peripheral blood of a patient who has been pretreated with ibrutinib or another interleukin-2 inducible T cell kinase (ITK) inhibitor and is resistant to treatment with ibrutinib or said another ITK inhibitor; b. culturing the PBMC in a culture containing a first culture medium having IL-2 and anti-CD3 / anti-CD28 antibodies for a period selected from the group consisting of 9, 10, 11, 12, 13, and 14 days, thereby performing an expanded culture of peripheral blood lymphocytes (PBL) from the PBMC; and c. recovering the PBL from the culture of step b, A method comprising.

2. The method according to claim 1, wherein in step b, the anti-CD3 / anti-CD28 antibody is bound to magnetic beads and the ratio of said beads to cells is 3:1 in the culture.

3. The method according to claim 1, wherein in step b, additional IL-2 is added to the culture on the 4th day of culturing the PBMC and the first culture medium is replaced in the culture.

4. The method according to claim 3, wherein the first culture medium is replaced with a second culture medium in the culture.

5. The method according to claim 4, wherein the first culture medium is different from the second culture medium.

6. The method according to claim 2, wherein the magnetic beads in step b are beads immobilized with an anti-CD3 / anti-CD28 antibody.

7. The method according to claim 1, wherein the peripheral blood is derived from peripheral blood of a patient suffering from a hematological malignancy.

8. The method according to claim 7, wherein the hematological malignancy is a liquid tumor.

9. The method according to claim 1, wherein the first culture medium contains 3000 IU / ml of IL-2. The culture is incubated at 37 °C, 5% CO ２ as claimed in claim 1, wherein the culture is incubated at 37 °C, 5% CO

10.

11. The method according to claim 1, wherein step b is carried out for 9 days.

12. The method according to claim 1, wherein step b is carried out for 11 days.

13. The method according to claim 1, wherein the patient has been pretreated with ibrutinib and is resistant to treatment with ibrutinib.

14. The method according to claim 1, wherein the patient has not been treated with ibrutinib or said another ITK inhibitor for at least 1 month prior to pretreatment with ibrutinib or another ITK inhibitor.

15. The method according to claim 1, wherein the patient has been pre-treated with ibrutinib or another ITK inhibitor for at least 3 months.

16. The method according to claim 1, wherein the patient has been pre-treated with ibrutinib for at least 3 months.

17. Peripheral blood lymphocytes (PBLs) for use in the treatment of hematological malignancies, wherein the PBLs are obtainable according to a method of expanding peripheral blood lymphocytes (PBLs) from peripheral blood, the method comprising a. obtaining a sample of peripheral blood mononuclear cells (PBMCs) from a patient with a hematological malignancy who has been pre-treated with ibrutinib or another interleukin-2 inducible T cell kinase (ITK) inhibitor and is resistant to treatment with ibrutinib or the other ITK inhibitor; b. culturing the PBMCs in a culture containing a first culture medium having IL-2 and anti-CD3 / anti-CD28 antibodies for a period selected from the group consisting of 9, 10, 11, 12, 13, and 14 days, thereby performing an expanded culture of peripheral blood lymphocytes (PBLs) from the PBMCs; and c. recovering the PBLs from the culture of step b, comprising peripheral blood lymphocytes (PBLs).

18. The peripheral blood lymphocytes (PBLs) according to claim 17, wherein in step b, the anti-CD3 / anti-CD28 antibody is bound to magnetic beads and the ratio of the beads to the cells is 3:1 in the culture.

19. The peripheral blood lymphocytes (PBLs) according to claim 17, wherein in step b, additional IL-2 is added to the culture on the 4th day of culturing the PBMCs and the first culture medium is replaced in the culture.

20. The peripheral blood lymphocytes (PBLs) according to claim 19, wherein the first culture medium is replaced with a second culture medium in the culture.

21. The peripheral blood lymphocytes (PBLs) according to claim 20, wherein the first culture medium is different from the second culture medium.

22. The peripheral blood lymphocytes (PBLs) according to claim 18, wherein the magnetic beads in step b are beads immobilized with an anti-CD3 / anti-CD28 antibody.

23. The peripheral blood lymphocytes (PBLs) according to claim 17, wherein the patient has been pre-treated with ibrutinib and is resistant to treatment with ibrutinib.

24. The peripheral blood lymphocytes (PBLs) according to claim 23, wherein the patient has been pre-treated with at least 3 rounds of an ibrutinib regimen.

25. The peripheral blood lymphocyte (PBL) according to claim 23, wherein the patient has been pre-treated with ibrutinib for at least 3 months.

26. The peripheral blood lymphocyte (PBL) according to claim 17, which has not been treated with ibrutinib or the other ITK inhibitor for at least 1 month prior to being pre-treated with ibrutinib or the other ITK inhibitor.

27. The peripheral blood lymphocyte (PBL) according to claim 17, wherein the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), activated B-cell (ABC) DLBCL, germinal center B-cell (GCB) DLBCL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, relapsed and / or refractory Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), mature B-ALL, Burkitt lymphoma, Waldenström macroglobulinemia (WM), multiple myeloma, myelodysplastic syndrome, myelofibrosis, chronic myeloid leukemia, follicular center lymphoma, indolent NHL, human immunodeficiency virus (HIV)-associated B-cell lymphoma, and Epstein-Barr virus (EBV)-associated B-cell lymphoma.

28. The peripheral blood lymphocyte (PBL) according to claim 17, wherein the hematological malignancy is a liquid tumor.

29. The peripheral blood lymphocyte (PBL) according to claim 22, wherein the liquid tumor is chronic lymphocytic leukemia or small lymphocytic lymphoma.

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